Carnosic acid attenuates lipopolysaccharide-induced liver injury in rats via fortifying cellular antioxidant defense system

Food and Chemical Toxicology 53 (2013) 1–9

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Carnosic acid attenuates lipopolysaccharide-induced liver injury in rats via fortifying cellular antioxidant defense system Qisen Xiang a, Zhigang Liu a, Yutang Wang a, Haifang Xiao a,b, Wanqiang Wu a, Chunxia Xiao a, Xuebo Liu a,⇑ a b

College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471003, China

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Article history: Received 26 July 2012 Accepted 1 November 2012 Available online 28 November 2012 Keywords: Carnosic acid Lipopolysaccharide Liver dysfunction Oxidative stress Inflammation

a b s t r a c t The study investigated the protective effects of carnosic acid (CA), the principal constituent of rosemary, on lipopolysaccharide (LPS)-induced oxidative/nitrosative stress and hepatotoxicity in rats. CA was administered orally to rats at doses of 15, 30 and 60 mg/kg body weight before LPS challenge (single intraperitoneal injection, 1 mg/kg body weight). The results revealed that CA inhibited LPS-induced liver damage and disorder of lipid metabolism, which were mainly evidenced by decreased serum levels of alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase. CA also inhibited LPSinduced oxidative/nitrosative stress by decreasing lipid peroxidation, protein carbonylation, and serum levels of nitric oxide. Histopathological examination demonstrated that CA could improve pathological abnormalities and reduce the immigration of inflammatory cells in liver tissues with LPS challenge. Concurrently, CA potently inhibited the LPS-induced rise in serum levels of the pro-inflammatory cytokines tumor necrosis factor-a and interleukin-6. CA supplementation markedly enhanced the body’s cellular antioxidant defense system by restoring the levels of superoxide dismutase, glutathione peroxidase, and glutathione in serum and liver after the LPS challenge. In conclusion, the present study suggests that CA successfully and dose dependently attenuates LPS-induced hepatotoxicity possibly by preventing cytotoxic effects of oxygen free radicals, NO and cytokines. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Lipopolysaccharide (LPS), also known as endotoxin, which is the major component of the outer membrane of Gram-negative bacteria, is known for its various biological and immunological activities. Research reports have shown that LPS plays a fundamental role in sepsis, systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction, including liver, lung, heart, gastrointestinal tract, and kidneys (Chen et al., 2007; Haghgoo et al., 1995; Yokozawa et al., 2003; Zhang et al., 2009). LPS administered to an organism is mainly cleared from the blood by the liver. Kupffer cells, the resident macrophages of the liver, are primarily responsible for this clearance. LPS can effectively activate Kupffer cells to generate an abundance of inflammatory substances, leading to the onset of liver damage. So the liver is the most sensitive organ to LPS-mediated adverse effects. In addition, during the LPS-related pathological course, a large number of mediators such as interleukins, tumor necrosis factor, reactive oxygen radicals, toxic eicosanoids, and platelet activating factor are released ⇑ Corresponding author at: College of Food Science and Engineering, Northwest A&F University, 28 Xi-nong Road, Yangling 712100, China. Tel./fax: +86 29 87092325. E-mail address: [email protected] (X. Liu). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.11.001

(Wolkow, 1998). Of them, reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to play major roles in the LPS-related pathological course (Cinel and Opal, 2009). Thus, the search for novel agents which are effective in inhibiting the production of reactive oxygen radicals is very important for the prevention and treatment of LPS-induced tissue injury. Recent research reports have shown that supplementation with natural antioxidants or plant extracts exert protective effects against LPS-induced various injury in vivo. For example, phytochemicals in Ginkgo biloba, Sun Ginseng, and blackberry, are potential to treat endotoxin-related diseases (Ilieva et al., 2004; Kang et al., 2007; Okoko and Ndoni, 2009; Sautebin et al., 2004; Watanabe et al., 2008). Rosemary (Rosmarinus officinalis L.) is a popular herb originated in the Mediterranean region, which is widely used as a culinary herb, flavouring agent, and naturally occurring antioxidant (Oberdieck, 2004). Recently, rosemary extracts garnered an additive E classification from the European Food Safety Authority and were approved as food antioxidants in Europe (Aguilar et al., 2008). Carnosic acid (CA, C20H28O4) is a phenolic diterpene isolated from various herbal plants including rosemary and sage (Salvia officinalis) (Schwarz and Ternes, 1992). CA has been widely reported to have the biological activities in mammals and mammalian cells including antioxidant (Schwarz and Ternes, 1992), antiobesity (Wang et al., 2011a,b), antitumor (Yesil-Celiktas et al.,

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2010), and neuroprotective effects (Azad et al., 2011). The CA showed anti-inflammatory effects in several in vitro assays using the human polymorphonuclear leukocytes and RAW 264.7 murine macrophage cell (Poeckel et al., 2008; Yu et al., 2009). In in vitro trials, CA also inhibited phorbol 12-myristate 13-acetate-induced ear inflammation in mice (Mengoni et al., 2011). Based on this observation, it was proposed that rosemary extracts or CA could present a new strategy to reduce endotoxin-related tissue injury. Nevertheless, there is no report about using CA or rosemary extract to ameliorate the LPS-induced oxidative stress and hepatotoxicity. In the present study, the protective effects of CA on LPS-induced toxicity in Sprague–Dawley rats (SD) were investigated. The antioxidative effects of CA on the LPS-induced inflammatory reaction in SD rats were elucidated by measuring in vivo levels of superoxide dismutase (SOD), nitric oxide (NO), glutathione (GSH), and cytokine in serum, as well as SOD, GSH, glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) in the liver. The effect of CA on the histopathology of liver tissue was also investigated.

2.3. Sample preparation for biochemical evaluation The animals were killed after the injections of 10% chloral hydrate (4 mL/ kg bw). Blood samples were centrifuged at 1400g at 4 °C for 10 min. The supernatants were used for the biochemical evaluation of serum. The livers were excised, weighed and kept for liver biochemical tests and histological assessment of hepatic pathology.

2.4. Biochemical evaluation of serum The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were determined using a Hitachi7180 automatic analyzer (Hitachi, Tokyo, Japan). The total SOD activity and GSH content were determined by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The amount of NO in serum was determined by measuring the stable NO metabolites, nitrate and nitrite, with a nitrite detection kit (Beyotime Institute of Biotechnology, Jiangsu, China) (Cai et al., 2007).

2.5. Assay of serum cytokines The serum IL-6 and TNF-a levels were measured using specific ELISA kits according to the manufacturer’s protocol (R&D Systems, Minneapolis, MN, USA).

2. Materials and methods 2.1. Chemicals and reagents LPS from Escherichia coli serotype 055:B5 (L2880) and rabbit anti-DNP polyclonal antibody (D9656), 2,4-dinitrophenylhydrazine (DNPH) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Mouse anti-b-actin (sc-47778) and goat antirabbit IgG-HRP (sc-2004) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The BCA protein assay kit and enhanced chemiluminescence (ECL) kit were obtained from Thermo Scientific (Rockford, IL, USA). Carnosic acid-rich rosemary extract (CRE) was obtained from Hainan Super Biotech Co. Ltd. (Haikou, Hainan, China), which was standardized to contain 71% CA. The total SOD, GSHPx, and GSH assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). PEG 400 were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). Dexamethasone (DEX) sodium phosphate injection (batch number: 11030211) was purchased from Tianjin Pharmaceutical Jiaozuo Co., Ltd. (Jiaozuo, Henan, China). Nitric oxide (NO) assay kit was purchased from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Enzyme-linked immunosorbent assay (ELISA) kits for rat tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6) were obtained from R&D Systems Inc. (Minneapolis, MN, USA).

2.2. Animal care and treatments A total of 30 male Sprague–Dawley rats, weighing 200–300 g, were provided by the Experimental Animal Center of Xi’an Jiaotong University (Xi’an, Shaanxi, China). All animals were housed in air-conditioned quarters with 12 h alternating light– dark cycle. The rats were fed a standard rat diet, provided with food and water ad libitum. This experiment was approved by the Northwest A&F University Animal Care and Use Committee (Yangling, Shaanxi, China), following the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rats were allowed to acclimatize to the laboratory conditions for 1 week before the start of the experiments. Then, the rats were randomly divided into six groups of five rats each as follows: Group I (Cont): the normal control group, the rats received normal saline. Group II (LPS): the model group, the rats received intraperitoneal injection of LPS. Group III (LPS + DEX): the LPS-stressed rats were treated with DEX. Group IV (LPS + CA15): the LPS-stressed rats were administered with low doses of CA. Group V (LPS + CA30): the LPS-stressed rats were administered with middle doses of CA. Group VI (LPS + CA60): the LPS-stressed rats were administered with high doses of CA. The rats in Group II–VI received intraperitoneal (i.p.) injection of LPS (1 mg/kg bw) every 5 days. The rats in Group II were injected i.p. with DEX (5 mg/kg bw) before the injection of LPS. CRE (equivalent to 15, 30 and 60 mg of carnosic acid) was dissolved in 3 mL 10% PEG 400 in normal saline (v/v). The rats in Group IV–VI were administered daily with low, middle and high doses of CRE, which were equivalent to 15, 30 and 60 mg CA/kg/day bw, respectively. The rats in the control group received no LPS but were given the equal-volume sterile saline and 10% PEG 400. The treatments were given for 35 days.

2.6. Biochemical evaluation of liver Briefly, liver was removed from all groups samples and were rinsed in isotonic saline solution. A 10% (w/v) liver homogenate in each case was prepared in 0.9% saline. Then, the homogenates were centrifuged at 3000 rpm for 10 min at 4 °C, and the protein content in the supernatant was measured using the BCA protein assay kit from Thermo Scientific (Rockford, IL, USA). An aliquot of the supernatant was used for the assay of SOD, glutathione peroxidase (GSH-Px), GSH, total nitric oxide synthase (tNOS) and inducible nitric oxide synthase (iNOS) by commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols.

2.7. Estimation of lipid peroxidation in serum and liver homogenate The quantitative measurement of lipid peroxidation in serum and liver homogenate were performed according to previously described methods (Todorova et al., 2005). The malondialdehyde (MDA) content, a measure of lipid peroxidation, was assayed in the form of thiobarbituric acid reacting substances (TBARS). In brief, 1.0 mL serum or 10% liver homogenates, 1.0 mL physiological solution and 1.0 mL 25% trichloracetic acid were added and the mixture was centrifuged at 2000 rpm for 20 min. After centrifugation, a 1.0 mL protein-free supernatant was mixed with 0.25 mL 1% thiobarbituric acid and the mixture was heated at 100 °C for 10 min. After cooling, the absorbance was measured at 532 nm. The results were expressed as nanomoles of MDA per milliliter serum or nanomoles of MDA per milligram of protein, using the molar extinction coefficient of TBARS (1.56  105 M 1 cm 1).

2.8. Estimation of protein carbonylation in liver homogenate The levels of protein carbonyl groups were determined by immunoblotting using an anti-DNP specific antibody. Briefly, protein samples were mixed with loading buffer and heated at 100 °C for 5 min. After being separated by SDS– PAGE electrophoresis, proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA) and derivated with DNPH (0.5 mM in 2 M HCl) for 5 min. The membranes were washed three times in 2 M HCl and five times in 100% methanol (5 min each). The membranes were blocked with 5% non-fat milk and incubated in a 1:2500 dilution of rabbit anti-DNP primary antibody (D9656; Sigma) at 4 °C overnight, followed by HRP-conjugated goat anti-rabbit IgG antibody (sc-2004, Santa Cruz) at room temperature for 2.5 h (Robinson et al., 1999; Xiang et al., 2012). Target proteins in the blots were visualized using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, USA), and visualized with the ChemiDoc XRS gel documentation system (Bio-Rad, Hercules, CA, USA).

2.9. Histological examination The liver specimens were obtained and promptly fixed in 10% phosphate-buffered formaldehyde for further studies. The specimens were embedded in paraffin, stained with hematoxylin and eosin (H&E), and examined using a Motic BA400 microscope (Motic Co., Ltd., Causeway Bay, HongKong, China).

Q. Xiang et al. / Food and Chemical Toxicology 53 (2013) 1–9 2.10. Statistical analysis All data were expressed as mean values ± standard deviation (SD). The intergroup variation was measured by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests. The level of statistical significance was established at p < 0.05. All statistical analyses were performed using the Statistical Package for Social Sciences version 16 (SPSS Inc., Chicago, USA).

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decreased total SOD activity and the amount of GSH in serum (p < 0.05) compared with the control group (Fig. 3). The results showed that serum SOD activity and GSH concentration in serum of the DEX-treated and CA-treated injured rats were significantly higher than those of model group rats (p < 0.05). Thus, CA prevented LPS-induced inflammatory reactions as indicated by decreased antioxidant defense systems.

3. Results 3.1. Effects of CA on LPS-induced lipid peroxidation and protein carbonylation MDA, an end product of lipid peroxidation, has been used as an index of oxidative stress. To examine the effect of CA administration on LPS-induced oxidative stress in rats, the MDA contents in serum and liver homogenate were measured by TBRAS assay. As shown in Fig. 1, LPS injection resulted in markedly increased production of MDA by 79.4% in serum and 101.8% in liver homogenate compared with the control group (p < 0.05). The treatment of DEX markedly blunted the rise of MDA levels in serum and liver. Compared with the LPS-treated group, the pretreatments of CA (5, 15 and 30 mg/kg bw) decreased the serum levels of MDA by 20.8%, 35.6%, and 42.0%, respectively (Fig. 1A). The treatment of CA also decreased the hepatic MDA contents by 12.7%, 27.6%, and 31.3%, respectively (Fig. 1B). These results showed that the administration of CA produced significant and dose-dependent attenuation in LPSinduced increase in lipid peroxidation. Carbonyl formation is an early and stable marker for protein oxidation. Protein carbonyl levels in the livers after the different treatments were determined by Western blot analysis with antiDNPH–protein adducts polyclonal antibody. The total amount of carbonylated proteins in the control rat livers was lower (Fig. 1C, lane 1). A significant increase in the protein carbonyl group content in the livers of LPS-treated rats was observed (Fig. 1C, lane 2). The administration of DEX and CA decreased the elevation of liver protein carbonyl content. The results also indicated that the inhibitory effect of CA was in a concentration-dependent manner (Fig. 1C, lanes 4–6). 3.2. Effects of CA on serum NO levels and activities of NOS isoforms in liver As shown in Fig. 2A, the LPS-treated rats showed a marked increase of NO levels in serum (104.5% increase, p < 0.05 versus control groups). When rats were injected i.p. with DEX (5 mg/kg bw), the serum NO levels were significantly decreased when compared with those of the LPS-treated rats (p < 0.05). Intragastric administration of CA significantly decreased the serum NO concentration in a dose-dependent manner. The effects of CA on activities of NOS isoforms in liver were also investigated. Compared with control groups, the activities of tNOS and iNOS in the livers of LPS-treated rats were increased by 36.7% and 97.1%, respectively (Fig. 2B and C). As shown in Fig. 2B and C, an i.p. injection of DEX could inhibit the increases of tNOS and iNOS activities in LPS-treated rats (p < 0.05). The administration of CA also significantly inhibited the increases of tNOS and iNOS activities in a dose-dependent manner (Fig. 2B and C). In contrast, the changes in constitutive NO synthase (cNOS) activities were not statistically significant (data not shown). 3.3. Effects of CA on LPS-induced changes in serum levels of SOD and GSH To assess the defense mechanisms involved in the LPS-induced oxidative stress, the total SOD activity and the levels of reduced GSH in serum were determined. The data showed that LPS potently

3.4. Effects of CA on LPS-induced changes in hepatic anti-oxidant profile As shown in Fig. 4, hepatic total SOD, GSH-Px activities and GSH content in LPS-treated rats were significantly lower than those in the control rats. The administration of DEX attenuated the decreased hepatic GSH content (p < 0.05), but had no effect on the changes of SOD and GSH-Px activities in liver. However, administration of CA increased the hepatic SOD, GSH-Px activities and GSH content in a dose-dependent way. There were no significant differences in the activities of SOD, GSH-Px and the amount of GSH between the control group and the LPS + CA60 group (p > 0.05). 3.5. Effects of CA on LPS-induced systemic inflammation Based on the facts that CA possesses anti-inflammatory effects and inflammatory cytokines play a key role in inflammation, we assumed that CA might have inhibitory effects on the production of pro-inflammatory cytokines. The effects of CA on LPS-induced changes in serum cytokines were shown in Fig. 5. LPS challenge caused marked increases in the levels of the pro-inflammatory cytokines TNF-a and IL-6 (p < 0.05) when compared with salinetreated controls. The administration of CA (30 or 60 mg/kg bw) prevented the elevation of TNF-a and IL-6 in serum (p < 0.05) in a dose-dependent way. In comparison, the pretreatment of DEX only inhibited the secretion of TNF-a, but had no effect on serum levels of IL-6 when compared with LPS-treated rats. 3.6. Effects of CA on LPS-induced liver dysfunction The results on the hepatoprotective effect of CA against LPS-induced acute liver damage in rats were shown in Fig. 6. The serum levels of ALT, AST, and ALP were significantly higher in LPS-treated rats than those in the saline control group (p < 0.05), indicating the severity of hepatic injury and cholestasis caused by LPS. Thus, compared with the saline control group, ALT, AST, and ALP in serum of LPS-treated rats were raised by 26.2%, 66.6%, and 59.2%, respectively. The activities of serum AST, ALT, and ALP were significantly reversed by treatment with CA in a dose-dependent manner. CA administered at 60 mg/kg bw significantly reduced the elevated plasma ALT by 21.2%, AST by 36.4%, and ALP by 40.2%. However, the pretreatment of DEX only inhibited the elevated plasma ALT and AST, but had no effect on ALP levels in the serum when compared with LPS-treated rats. 3.7. Histological changes in liver tissue Under light microscope, the control animals showed normal hepatocytes with well preserved cytoplasm, prominent nucleus and nucleolus. There was no notable of morphological alterations, inflammation or necrosis in these animals (Fig. 7A). In contrast, livers of LPS-administered rats showed hepatic lobular structure to be distorted and marked morphological disruption such as lymphoplasmacytic infiltration, cytoplasmic vacuoles, and ballooning degeneration of hepatocytes (Fig. 7B). Administration of DEX before LPS challenge resulted in significant morphological protection against LPS induced liver damage (Fig. 7C). The administration of

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LPS Fig. 1. Effects of CA on LPS-induced oxidative stress in rats. MDA levels in serum (A) and liver homogenate (B) were measured by TBARS method as described in Section 2. Each value represents the mean ± SD (n = 5). An ANOVA with Duncan’s multiple-range test (p < 0.05) was used for statistical analysis. (C) Carbonylated proteins in the liver were determined by western blot procedure as described in Section 2. b-Actin was used as an internal control to verify equal protein loading.

low doses of CA did not obviously change histological appearances of livers compared with the LPS model group (Fig. 7D). However, middle- and high-dose supplementation with CA inhibited the pathological change of liver injury (Fig. 7E and F). The liver tissue of rats in Group VI displayed cell recovery compared with rats induced with LPS alone (Fig. 7F).

4. Discussion LPS is a bacterial endotoxin, and induces extensive damage to various organs including heart, gastrointestinal tract, kidney, liver, brain, and lung (Chen et al., 2007; Haghgoo et al., 1995; Nolan et al., 2003; Yang et al., 2012; Yokozawa et al., 2003; Zhang et al., 2009). The use of antioxidants and drugs from herbal origins has been shown to be beneficial in reversing the LPS-induced oxidative stress and hepatotoxicity (Ilieva et al., 2004; Kang et al., 2007; Sautebin et al., 2004; Watanabe et al., 2008). In the present study, the inhibitory effect of CA supplementation on LPS-induced oxidative stress and hepatotoxicity in SD rats was investigated. Based on the 90-day feeding studies in rats with different rosemary extracts, the no observed adverse effect level (NOAEL) is 20–60 mg/ kg bw/day of carnosol plus CA (Aguilar et al., 2008). So an initial dose of 60 mg/kg bw was chosen as the highest dose of CA. Lipid peroxidation is a common event in toxic phenomenon (Requintina and Oxenkrug, 2003). MDA and protein carbonyl, as markers of lipid and protein oxidation, were analysed to evaluate the protective effect of CA on LPS-induced oxidative stress in SD

rats. Concordant with previous observation (Lu et al., 2005), we reported in the present study that the administration of LPS caused significant oxidative injury, indicated as significant increases in lipid peroxidation and protein carbonylation. Our data also showed that supplementation with CA significantly and dose-dependently inhibited lipid peroxidation and the formation of protein carbonyl groups. All organisms possess well-developed systems of antioxidant defense to maintain the cellular redox state, including both antioxidant enzymes and non-enzymatic antioxidants. SOD and GSH-Px are the major antioxidant enzymes, which can help to protect the body from oxidative damage. GSH, manufactured mainly in the liver, is the most abundant and important endogenous antioxidant with particularly high concentrations in the liver. GSH also is the liver’s major detoxifying agent, enabling the body to get rid of toxic compounds and heavy metals. In this study, GSH, SOD, and GSH-Px levels significantly decreased in the serum and liver of LPStreated rats, indicating that the antioxidant system changed significantly after LPS infection. All of these found perturbations in the antioxidant system were restored by the administration of CA. Our results are consistent with the those of Sahu et al. (2011), who find CA can enhance the levels of GSH, SOD, GSH-Px, and catalase in cisplatin-treated rats. This study showed that increasing antioxidant activity in the body is probably one of the ways for CA to prevent oxidative damage and hepatic injury caused by LPS. ALT and AST are the most common liver transaminases. AST is present in both the cytoplasm and mitochondria of hepatocytes, whereas ALT is present only in the cytoplasm. ALP is an enzyme that transports metabolites across cell membranes. Hepatic ALP

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Fig. 3. Effects of CA on serum levels of SOD and GSH. The serum levels of SOD (A) and GSH (B) were measured by using commercial kits as described in Section 2. Each value represents the mean ± SD (n = 5). An ANOVA with Duncan’s multiple-range test (p < 0.05) was used for statistical analysis.

is found histochemically in the microvilli of bile canaliculi and on the sinusoidal surface of hepatocytes (Giannini et al., 2005). An increase in serum ALP levels is frequently associated with a variety of diseases, such as extrahepatic bile obstruction, intrahepatic cholestasis, infiltrative liver disease, and hepatitis (Wiwanitkit, 2001). The marked rises in serum levels of ALT, AST, and ALP, due to the damage to the plasma and disturbance in the transport functions of hepatocytes, are circulating markers of LPS-induced hepatocyte injury. In the present investigation, LPS caused marked increases in the serum levels of ALT, AST, and ALP, which is in agreement with previous reports (Kim and Kim, 2002). The protective effect of CA may be due to the antioxidant effect of CA on LPS-induced oxidative stress. LPS is a potent stimulator of nitric oxide (NO), which is critical for the LPS-induced oxidative/nitrosative stress and tissue injury

(Tsuji et al., 2005). Thiemermann et al. reported that treatment of endotoxaemia for 6 h resulted in a 4.5-fold rise in the serum levels of nitrite, a stable end product of NO metabolism (Thiemermann et al., 1995). The increased levels of NO after endotoxin challenge can combine with superoxide anion (O2 ), leading to formation of the peroxynitrite anion (ONOO ), which is a longlived cytotoxic oxidant and capable of resulting in tissue injury and organ dysfunction (Beckman et al., 1990; Pacher et al., 2007). Furthermore, peroxynitrite anion can stimulate the production of TNF-a and IL-6 in human monocytes via activation of oxidant-sensitive nuclear factor kappa B (NF-jB) (Matata and Galiñanes, 2002). NO also stimulates hydrogen peroxide (H2O2) and O2 production by mitochondria (Poderoso et al., 1996), which can cause oxidative damage to multiple tissues. LPS can also induce iNOS expression in Kupffer cells and hepatocytes (Zhang et al., 2000). LPS-induced

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Fig. 5. Effects of CA on production of proinflammatory cytokines from LPS-treated rats. Serum levels of TNF-a (A) and IL-6 (B) were measured using ELISA kits. Each value represents the mean ± SD (n = 5). Significant differences among groups were determined by ANOVA, followed by Duncan’s multiple-range test at a significance level of p < 0.05.

iNOS expression is suggested to be the main source of NO during inflammatory reaction and many diseases are caused by overproduction of NO. Very recently, Kuo et al. have studied that supercritical carbon dioxide extract from R. officinalis leaves and CA markedly suppressed the LPS-induced production of NO and the expression of iNOS in RAW 264.7 macrophage cells (Kuo et al., 2011). The activities of total NOS and iNOS in liver were assayed in this study. Our study showed that serum NO content and iNOS activity in liver were significantly elevated after LPS challenge in rats and the LPS-induced nitrosative stress was significantly and dose-dependently attenuated by the administration of CA. A large number of cytokines produced by activated macrophages and neutrophils are intimately involved in many immune

and inflammatory responses elicited by LPS (Andreasen et al., 2008; Beutler and Cerami, 1988; Steib, 2011). The Kupffer cells, the resident macrophages of the liver, account for approximately 10–15% of the total liver cell population and constitute 80–90% of the tissue macrophages in the reticuloendothelial system (Andreasen et al., 2008; Steib, 2011). Kupffer cells are capable of releasing large amounts of proinflammatory cytokines such as TNF-a and IL-6 after stimulation with LPS (Chiao et al., 2005). Among the cytokines produced in the liver during inflammation, TNF-a and IL-6 are particularly important because of their many biological effects both in the liver and elsewhere. TNF-a can activate a range of transduction pathways such as NF-jB or Janus kinase (JAK) pathways to induce hepatocyte death (Nakagawa and

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LPS Fig. 6. Effects of CA on liver enzymes parameters of LPS-treated rats. The serum levels of ALT (A), AST (B), and ALP (C) were determined using an automated biochemical analyzer Hitachi-7180 (Hitachi, Tokyo, Japan). Each value represents the mean ± SD (n = 5). An ANOVA with Duncan’s multiple-range test (p < 0.05) was used for statistical analysis.

Fig. 7. Hematoxylin and eosin staining of liver sections (100). Hepatic photomicrographs of representative rat are shown from each of the six groups. (A) Control liver tissue without any treatment; (B) liver tissue of rats treated with LPS alone; (C) liver tissue of rats in Group III (LPS + DEX); (D) liver tissue of rats in Group IV (LPS + CA15); (E) liver tissue of rats in Group V (LPS + CA30); (F) liver tissue of rats in Group VI (LPS + CA60).

Maeda, 2012). The intracellular signaling of IL-6 is triggered by glycoprotein 130 (gp130)-associated JAK kinases and signaled through signal transducer and activator of transcription 3 (STAT3),

mitogenactivated protein kinase (MAPK) and PI3K pathways. Especially, the pro-inflammatory effect of hepatocyte STAT3 is likely mediated via its induction of acute phase proteins and chemokines

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in the liver (Eulenfeld et al., 2012; Wang et al., 2011a,b). Previous research reported that CA markedly suppressed the LPS-induced production of TNF-a, IL-1b, and IL-6 in vitro (Kuo et al., 2011; Yanagitai et al., 2012). In this study, we also observed an abrupt increase in serum TNF-a and IL-6 levels after LPS challenge in vivo. Previous studies showed that CA strongly suppressed the production of NO, TNF-a, and IL-6, IL-8 by blocking the NF-jB and its upstream signaling including Syk/Src, phosphoinositide 3-kinase (PI3K), Akt, inhibitor of jBa (IjBa) kinase (IKK), and IjBa for NF-jB activation (Oh et al., 2012). In addition, it has been also reported that CA is able to induce Keap1/Nrf2 pathway (Satoh et al., 2008), which is a typical cellular defensive system against cellular oxidative stress and participates in cellular anti-inflammatory responses (Kaspar et al., 2009). So, further study is needed to clarify the role of CA-regulated NF-jB signalling pathway and Keap1/Nrf2 pathway in the protective effects of CA on LPS-induced oxidative stress and liver injury in rats. Dexamethasone (DEX), a synthetic member of the glucocorticoid class of hormones, acts as an anti-inflammatory and immunosuppressant. In particular, a recent study has reported that DEX can attenuate LPS-induced liver injury, by downregulating glucocorticoid-induced tumor necrosis factor receptor ligand in Kupffer cells (Wei et al., 2011). Thus, DEX was used as a positive control in this study. In consistent with the results of previous reports (Wei et al., 2011), DEX markedly inhibited LPS-induced oxidative injury and liver dysfunctions in this study. 5. Conclusions In conclusion, the present data suggest that CA supplementation can provide protection against LPS-induced oxidative injury and liver toxicity, which is possibly at least in part by its antiinflammatory and increasing the antioxidant defense mechanism in rats. It is inferred that CA and rosemary may be used to attenuate oxidative/nitrosative stress and liver dysfunctions. However, further studies are still needed to elucidate possible molecular mechanisms underlying the protective effects of CA. Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgements This research was supported by the Program for New Century Excellent Talents in University (NCET), Chinese Ministry of Education. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fct.2012.11.001. References Aguilar, F., Autrup, H., Barlow, S., 2008. Use of rosemary exracts as a food additive. Scientific opinion of the panel on food additives, flavourings, processing aids and materials in contact with food. EFSA J. 721, 1–29. Andreasen, A.S., Krabbe, K.S., Krogh-Madsen, R., Taudorf, S., Pedersen, B.K., Moller, K., 2008. Human Endotoxemia as a model of systemic inflammation. Curr. Med. Chem. 15, 1697–1705. Azad, N., Rasoolijazi, H., Joghataie, M.T., Soleimani, S., 2011. Neuroprotective effects of carnosic acid in an experimental model of Alzheimer’s disease in rats. Cell J. 13, 39–44. Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A., Freeman, B.A., 1990. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87, 1620– 1624.

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