Chemico-Biological Interactions 213 (2014) 51–59
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Protective effects of neohesperidin dihydrochalcone against carbon tetrachloride-induced oxidative damage in vivo and in vitro Lihua Hu a, Lingrui Li a, Demei Xu a, Xiaomin Xia a, Ruxian Pi b, Duo Xu a, Wenchao Wang a, Hong Du a, Erqun Song a, Yang Song a,⇑ a Key Laboratory of Luminescence and Real-Time Analytical Chemistry, Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, People’s Republic of China b Department of Hepatobiliry Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 6 November 2013 Received in revised form 15 January 2014 Accepted 5 February 2014 Available online 14 February 2014 Keywords: Hepatotoxicity Antioxidant Liver injury COX-2 iNOS HepG2 cell
a b s t r a c t The purpose of this study was to investigate the possible hepatoprotective effects of neohesperidin dihydrochalcone (NHDC) on carbon tetrachloride (CCl4)-induced acute oxidative injury in vivo and in vitro. In a mouse model, intraperitoneal injection of CCl4 resulted in a significant increase in serum aspartate transaminase (AST) and alanine transaminase (ALT) activities. Histopathological examination revealed severe hepatocyte necrosis and destruction of architecture in liver lesions, and immunohistochemical staining illustrated a remarkable enhancement of COX-2 and iNOS expression. The levels of hepatic antioxidant, such as, catalase (CAT), total superoxide dismutase (T-SOD), glutathione peroxidase (GP-X) and glutathione (GSH) were decreased, compared to the control group. However, pretreatment of NHDC for six consecutive days significantly ameliorated these changes. Moreover, Western blotting assay indicated pretreatment with NHDC also down-regulated CCl4-induced protein expressions of NF-jB, IL-6, caspase 3 and caspase 8. In HepG2 cell model, CCl4-treatment caused significant decrease in cell viability, antioxidant activities and GSH level, increase in intracellular reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) level. Interestingly, pretreatment of NHDC effectively relieved CCl4-induced oxidative damage in a dose-dependent manner. In conclusion, NHDC appeared to possess promising anti-oxidative and anti-inflammatory capacities, it is possible to be used as a hepatoprotective agent. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Free radicals and oxidative stress play pivotal roles in the pathogenesis of a number of human diseases, such as cardiovascular diseases and diabetes, which led to the enthusiastic use of antioxidants in the treatment and prevention of these diseases in clinic [1,2]. In general, flavonoids possess high antioxidant Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CAT, catalase; CCl4, carbon tetrachloride; COX-2, cyclooxygenase-2; DAB, 3,30 -diaminobenzidine tetrahydrochloride; DCFH-DA, 20 ,70 -dichlorodihydrofluorescein diacetate; GP-X, glutathione peroxidase; GSH, glutathione; HepG2, human hepatoma cell line; IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; NF-jB, nuclear factor-kappa B; NHDC, neohesperidin dihydrochalcone; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; T-SOD, total superoxide dismutase. ⇑ Corresponding author. Present address: College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing 400715, People’s Republic of China. Tel.: +86 23 68251503; fax: +86 23 68251225. E-mail addresses: [email protected], [email protected] (Y. Song). http://dx.doi.org/10.1016/j.cbi.2014.02.003 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
activities directed toward O 2 , HO and alkoxyl radicals [3–5]. Neohesperidin dihydrochalcone (NHDC, 3,5-dihydroxy-4-(3-hydroxy-4-methoxyhydrocinnamoyl)phenyl 2-O-b-L-rhamnopyranosyl-b-D-glucopyranoside, structures presented in Fig. 1) belongs to a family of bycyclic flavonoids dihydrochalcones, which have two benzenoid rings joined by a three-carbon bridge. NHDC is manufactured by catalytic hydrogenation of neohesperidin in industry [6]. Essentially, NHDC is the ring-opening product of neohesperidin. NHDC is approximately 1500 times sweeter than sucrose [7], which has been used in food, beverage and pharmaceutical as an intense, low caloric artificial sweetener for half a century [8,9]. NHDC is well tolerated and is not associated with any particular toxicity as a well-known sweetener. No adverse effects were observed at NHDC levels of up to 5% of the diet of Wistar rats in embryotoxicity and teratogenicity studies [6]. The mutagenicity of NHDC was assessed in different experimental models, and no mutagenic activity was detected [10–13]. These results encouraged
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Three hours after final NHDC treatment, mice were injected with CCl4 (10 mL/kg body weight, v/v = 1:49 in olive oil). Twenty-four hours after the last dose of injection, mice were anesthetized with CO2. Blood samples were collected by cardiac puncture and allowed to clot for 45 min at room temperature. The livers were excised careful, washed twice with saline, blotted dry on a filter paper, weighed, then cut into two pieces. One halves were used for histopathological and immunohistochemical analysis. Other halves were used for hepatic homogenate preparation. Fig. 1. Chemical structures of NHDC.
us to investigate the antioxidant activity of NHDC. In recent studies, NHDC showed outstanding radical scavenging activity against ABTS+, O 2 , HO , H2O2 and HOCl [14,15]. In another study, NHDC is more effective in the inhibition of HOCl-induced protein degradation, plasmid DNA strand cleavage and cell death than ascorbic acid, mannitol and butylated hydroxy toluene [16]. However, to the best of our knowledge, the antioxidant activity of NHDC towards CCl4-induced acute hepatic injury has never been explored in vivo or in vitro. In the present study, mice and HepG2 cell models were employed to investigate the protective effects and mechanism of NHDC on carbon tetrachloride (CCl4)-induced acute liver injury.
2.3. Biochemical parameters of liver function Serums were separated by centrifugation of blood at 600g for 15 min. Serum AST and ALT activities were measured by diagnostic kits (Nanjing Jiancheng Institute of Biotechnology). 2.4. Histopathological examination Halves of liver were fixed for 24 h in 10% buffered formalin. They were embedded in paraffin and sectioned for 5 lm thickness. After hematoxylin–eosin (H&E) staining, slides were observed for histopathological changes using Nikon TE2000 fluorescence microscope (Nikon, Japan). Representative images were presented.
2. Materials and methods
2.5. Immunohistochemical staining
2.1. Materials
The paraffin-embedded sections were deparaffinized and rehydrated. Antigen retrieval was performed with a 1 mM EDTA buffer (pH = 9.0) in a microwave for 3 min. Then, the following steps were performed according to the instructions of Histostain™-plus and DAB substrate kits. Briefly, 3% H2O2 was used to block endogenous peroxidase activity for 20 min and non-specific protein binding was blocked by normal goat serum for 20 min. The treated slides were incubated in a moist box at 4 °C overnight with rabbit iNOS and COX-2 antibodies (1:100, dilution) followed by incubation biotin-labeled goat anti-rabbit IgG and horseradish peroxidaseconjugated streptavidin for 50 min, respectively. DAB fluid including 1 mL distilled water, 50 lL H2O2 (20) and 50 lL DAB (20) was used in color development and counterstained by hematoxylin. Images were taken by a light microscopy (magnification, 100, Nikon Eclipse Ti-SR).
NHDC (CAS number, 20702-77-6), CCl4, 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) and olive oil were purchased from Aladdin Reagent Database Inc. (Chengdu, China). Diagnostic kits used for the determination of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GP-X), aspartate transaminase (AST), alanine transaminase (ALT), reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Histostain™-plus kit and 3,30 -diaminobenzidine tetrahydrochloride (DAB) substrate kit were provided by Zhongshan Golden Bridge Biotechnology (Beijing, China). Inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and b-actin antibodies were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Interleukin-6 (IL-6), nuclear factor-kappa B (NF-jB), caspase 3 and caspase 8 antibodies were purchased from Proteintech Group Inc. (Wuhan, China). All other chemicals used were of highest commercial grade. 2.2. Animals, treatment and tissue collection The animal experiments were performed in accordance with the guideline of the Animal Care Committee of Southwest University. Male Kunming mice (22 ± 2 g) were purchased from Chongqing Tengxin Biotechnology Co. Animals were maintained under standard conditions of humidity (50%), temperature (25 ± 2 °C) in a 12 h light and 12 h dark cycle. They were fed standard rodent chow and had free access to water, acclimatized for at least one week prior to use. Mice were assigned to four groups randomly with 6 mice in each group: (1) control group, administered appropriate vehicles throughout; (2) NHDC group, received at a daily dose of NHDC (100 mg/kg body weight, dissolved in a 8% Tween 80/H2O vehicle and prepared before use) by oral gavage for 6 consecutive days; (3) CCl4 group, received saline once daily for 6 consecutive days. Three hours after final saline treatment, mice were intraperitoneally injected with CCl4 (10 mL/kg body weight, v/v = 1:49 in olive oil); (4) NHDC + CCl4 group, received at a daily dose of NHDC (100 mg/kg body weight) by oral gavage for 6 consecutive days.
2.6. Hepatic homogenate preparation Half livers were rinsed in ice-cold physiological saline and homogenized in Tris–HCl buffer (0.01 M, pH = 7.4) to give a 10% homogenates. Homogenates were centrifuged at 3000 rpm, 4 °C for 10 min and supernatants were collected for catalase (CAT), total superoxide dismutase (T-SOD) and glutathione peroxidase (GP-X) activities and reduced glutathione (GSH) level measurement. 2.7. Cell culture and treatment The human hepatoma cell line (HepG2) was obtained from Third Military Medical University, Chongqing, China. Cells were grown on RPMI 1640 medium with 10% fetal bovine serum (HyClone, USA), antibiotics (100 U/mL penicillin, 100 lg/mL streptomycin) at 37 °C and 5% CO2. Exponential growth phase cells were transferred onto 96-well culture plates at 1 104 cells/well and permitted to adhere overnight at 37 °C. Cells were treated with NHDC (10, 20 or 30 lM) or vehicle for 1 h, then, medium was replaced with fresh medium containing 0.5% (v/v) CCl4 for 24 h. Following incubation, cells were lysed with 300 lL of 0.1% Triton X-100 on ice for 10 min and sonicated for 10 s. Homogenates were centrifuged at 12,000 rpm, 4 °C for 20 min, and supernatants were collected for further investigation.
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2.8. Cell viability assay
by measuring the concentration of red adduct (absorbance at 532 nm) according to the methods of Heath and Parker [18].
Cell viability was evaluated using the MTT assay [17]. After incubation with CCl4 for 3 h, cells were treated with MTT (final concentration of 5 mg/mL) for 4 h at 37 °C. The MTT-containing medium was removed and 100 lL of DMSO were added to all wells, mixed thoroughly with a 10 min shake. The optical density (OD) of each well was measured at 490 nm using a microplate reader (BioTek ELX800). 2.9. Determination of reactive oxygen species (ROS) generation ROS level in HepG2 cells was determined using DCFH-DA as fluorescent probe. Fluorescence was recorded with an excitation wavelength of 488 nm and an emission wavelength of 526 nm (F7000, HITACHI). The fluorescence images were captured with a fluorescence microscope (OLYMPUS IX71) in order to obtain comparable data. 2.10. Determination of lipid peroxidation The level of TBARS was measured by commercial kits according to the manufacturer’s instruction (Nanjing Jiancheng Institute of Biotechnology). In brief, the formation of TBARS was determined
Table 1 Effect of NHDC on serum biochemical parameters in 0.5% CCl4-intoxicated mice. Data represent the mean ± SEM of three independent experiments. Significant differences are indicated by ⁄⁄⁄p < 0.001 as compared with control group, ##p < 0.01 and #p < 0.05 as compared with CCl4 group. Groups
Treatment
AST (IU/L)
ALT (IU/L)
Group Group Group Group
Control NHDC CCl4 NHDC + CCl4
54.8 ± 20.76 32.84 ± 15.79 184.44 ± 36.48⁄⁄ 56.36 ± 32.85##
17.52 ± 1.97 18.54 ± 1.83 329.92 ± 3.49⁄⁄⁄ 315.53 ± 7.05#
I II III IV
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2.11. Determination of antioxidants Levels of T-SOD, CAT, GP-X and GSH in HepG2 cells (or liver homogenates) were measured by commercial kits according to the manufacturer’s instructions (Nanjing Jiancheng Institute of Biotechnology). T-SOD activity was determined based on the reaction of SOD and nitrotetrazolium blue chloride, the product was measured with spectrophotometer at its maximum absorbance of 550 nm [19]. CAT activity was determined by measuring the H2O2 decomposition (absorbance at 240 nm) [20]. GP-X activity was measured by following the absorbance at 412 nm based on the oxidation of NADPH with H2O2 [21]. GSH level was measured by using the method of Anderson, the colorimetric reaction was monitored at 405 nm spectrophotometrically as 5,50 -dithiobis-2nitrobenzoic acid (DTNB) was reduced into 2-nitro-5-thiobenzoic acid (TNB) by GSH [22].
2.12. Western blotting Briefly, proteins were extracted from liver, separated by electrophoresis in 10% SDS–PAGE and transferred onto nitrocellulose membrane. After blocking with 10% skimmed milk, membranes were incubated with primary antibodies of rabbit polyclonal antibodies IL-6, NF-jB, caspase 3 and caspase 8 at room temperature for 4 h. After washing, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). Finally, immune-reactive protein bands were detected by the HRP substrate DAB system. Representative blots were chosen from three independent experiments. Densitometric analysis of western blots was performed with the use of ImageJ software. Protein levels were standardized by comparison with b-actin.
Fig. 2. Effect of NHDC on liver histopathology changes stained with hematoxylin–eosin. (A) Control mice, (B) mice treated by NHDC at 100 mg/kg, (C) CCl4-intoxicated mice and (D) mice treated by NHDC at 100 mg/kg and CCl4. Typical examples were shown from three independent experiments. Original magnification: 400x. The arrows show necrotic area.
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2.13. Statistical analysis
3. Results
All experiments were performed with three times independently. Data were presented as means ± SEM. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by least significance difference (LSD) multiple comparison tests using SPSS 18.0 software. p < 0.05 was considered to be significant.
3.1. Effect of NHDC on serum AST and ALT activities Serum AST and ALT are two common biomarkers of liver damage. The effect of NHDC on AST and ALT activities in serum were shown in Table 1. In the control group, AST and ALT activities were 54.8 ± 20.76 and 17.52 ± 1.97 IU/L, respectively. Pretreatment with
Fig. 3. Effect of NHDC on hepatic (A) COX-2 and (B) iNOS expression in CCl4-intoxicated mice. Typical examples were shown from three independent experiments. Original magnification: 100. The arrows show positive staining.
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NHDC alone has no significant effect on AST and ALT activities. However, Serum AST and ALT activities were significantly (p < 0.001) elevated after CCl4 injection, as compared to the control group. Surprisingly, pretreatment with NHDC at dose of 100 mg/ kg/day significantly decreased AST and ALT activities as compared to CCl4 group, p < 0.01 and p < 0.05, respectively. 3.2. Effect of NHDC on CCl4-induced hepatic necrosis In Fig. 2, the effect of NHDC on CCl4-induced hepatic histopathological damage was presented. Intraperitoneal injection of CCl4 resulted in histological changes in liver morphology, including massive cell necrosis and loss of hepatocyte architecture around the blood vessels (Fig. 2C). However, NHDC extensively alleviated CCl4-induced hepatic histopathological damage (Fig. 2D).
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in CCl4 group, compared to the control group (p < 0.05) (Fig. 4D). However, pretreatment with NHDC significantly elevated GSH levels in liver tissues compared to CCl4 group (p < 0.05).
3.5. Effect of NHDC on HepG2 cell viability The protective role of NHDC was further evaluated in human hepatocellular carcinoma HepG2 cells. In Fig. 5, MTT results showed that CCl4 caused significant loss of cell viability (about 20%), compare to the control group, p < 0.01. Interestingly, preincubation of NHDC in CCl4-intoxicated HepG2 cells increased cell viability in a dose-dependent manner, significant difference was found in the group of 30 lM NHDC (p < 0.05 compared to CCl4 group).
3.3. Effect of NHDC on CCl4-induced inflammatory response COX-2 and iNOS are two major inflammatory mediators implicated in inflammation. Immunohistochemical analysis revealed considerable up-regulation of hepatic COX-2 and iNOS proteins after CCl4 injection (shown as brown-colored stain), whereas the levels of COX-2 and iNOS were low in the control group (Fig. 3). However, NHDC attenuated the expression of proteins COX-2 and iNOS considerably. 3.4. Effect of NHDC on hepatic antioxidants in mice CCl4-intoxication resulted in a significant decrease in hepatic CAT, T-SOD and GP-X activities compared to the control group, p < 0.05 (Fig. 4A–C). On the contrary, pretreatment with NHDC restored these hepatic antioxidant enzyme activities. GSH, a sensitive marker of oxidative stress, was detected to further evaluate the degree of oxidative injury. Hepatic GSH level significantly decreased
Fig. 5. Effect of NHDC on 0.5% CCl4-treated HepG2 cell viability. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄p < 0.05 as compared to control group, #p < 0.05 as compared to CCl4 group.
Fig. 4. Effect of NHDC on hepatic (A) T-SOD, (B) CAT and (C) GP-X activities and (D) GSH level in CCl4-intoxicated mice. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01 and ⁄p < 0.05 as compared with control group, ###p < 0.001, ##p < 0.01 and #p < 0.05 as compared to CCl4 group.
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3.6. Effect of NHDC on ROS generation in HepG2 cells Intracellular ROS production plays an important role in CCl4induced oxidative damage. ROS level was investigated using DCFH-DA as a fluorescence probe. We observed that CCl4 intoxication caused a significant increase in ROS production (p < 0.05) (Fig. 6A). However, ROS level was inhibited by NHDC significantly in the 30 lM NHDC group (p < 0.05), compared to CCl4 group. Furthermore, by applying fluorescence microscopy, it was demonstrated that ROS level was considerably elevated in HepG2 cells by the treatment of CCl4 compared to unstimulated cells. Consistently, ROS was decreased by the pretreatment of NHDC in dose-dependent manner (Fig. 6B).
3.7. Effect of NHDC on lipid peroxidation in HepG2 cells Thiobarbituric acid reactive substances (TBARS) are formed as a byproduct of lipid peroxidation. After treatment with CCl4, TBARS level increased by about 2-fold (Fig. 7). However, pretreatment with NHDC inhibited TBARS in HepG2 cells dose-dependently, 20 and 30 lM NHDC groups showed statistical significance, p < 0.01 against CCl4 group.
Fig. 7. Effect of NHDC on TBARS level in 0.5% CCl4-treated HepG2 cells. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄⁄p < 0.01 as compared with control group, ##p < 0.01 as compared with CCl4 group.
3.8. Effect of NHDC on antioxidants in HepG2 cells Intracellular levels of enzymatic and non-enzymatic antioxidants were shown in Fig. 8. CCl4-intoxicated HepG2 cells showed
Fig. 6. Effect of NHDC against 0.5% CCl4-induced ROS formation in HepG2 cells. (A) ROS level was presented by the fluorescent product of DCFH-DA. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄p < 0.05 as compared with control group, #p < 0.05 as compared with CCl4 group. (B) Cells were stained with DCFH-DA probe and imaged by fluorescence microscope.
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a significant decrease in CAT, T-SOD and GP-X activities and GSH content (p < 0.05 or p < 0.01 against the control group). Pretreatment with NHDC elevated these antioxidants dose-dependently. 3.9. Effect of NHDC on the protein expression in liver tissue Western blot analysis showed that the levels of IL-6, NF-jB, caspase 3 (cleaved form) and caspase 8 proteins were observably up-regulated in CCl4 group, which was subsequently reduced under treatment with NHDC (Fig. 9). 4. Discussion Flavonoids are ubiquitous polyphenolic compounds existing in plants, categorized into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones [23]. It is well known that one of the benefits of flavonoids is protection against cardiovascular disease and oxidative stress as antioxidants. [24]. Besides direct scavenging of free radicals, flavonoids can prevent tissue injury by activation of antioxidant enzymes [25], chelating of metal ion [26], reduction of a-tocopheryl radicals [27], inhibition of oxidases [28], mitigation of nitric oxide [29], increasing of uric acid levels [30], etc. As a group of minor flavonoids, dihydrochalcones have not been widely investigated for their strength of antioxidant activity. Interestingly, Dziedzic et al. demonstrated that dihydrochalcones have higher antioxidant capacity than corresponding chalcones and flavanones [31]. CCl4 causes severe liver damage with necrotic and apoptotic hepatocellular injury. In the principle of liver injury, oxidative stress resulting from CCl4 metabolism plays an important role. CCl4 was metabolized into trichloromethyl radical (CCl3 ) by the cytochrome p450, then reacts with oxygen to form trichloromethylperoxy
Fig. 9. Effects of NHDC on the protein expressions in CCl4-intoxicated mice. Protein expressions of IL-6, NF-jB, caspase 3 and caspase 8 were assessed by Western blotting. The particular procedure was performed as described in Material and Methods section and protein expressions were analyzed by SDS–PAGE electrophoresis. The house keeping gene, b-actin, was used as control. One of three representative experiments is shown.
radical (CCl3OO), resulting in protein and DNA damage and lipid peroxidation [32]. Therefore, diminishing free radical directly and retarding the propagation of oxidative chain reaction are practicable strategies to preventing CCl4-induced hepatotoxicity. CCl4-induced hepatic injury is a widely used experimental model in screening of hepatoprotective drugs [33]. The present study showed that NHDC possesses a hepatoprotective effect against CCl4-induced damage as evidenced by significant histopathological alterations, restoring antioxidant enzyme activities and reducing inflammation and apoptosis compared to CCl4treated mice in the absence of NHDC. According to previous study, CCl4-treatment altered membrane integrity and increased serum AST and ALT activities [34]. It is well-accepted that antioxidants reducing serum AST and ALT activities exert hepatoprotective activity, our results suggested a promising potential of NHDC on preventing CCl4-induced hepatotoxicity, Table 1.
Fig. 8. Effect of NHDC on (A) total SOD (T-SOD), (B) CAT and (C) GP-X activities and (D) GSH level in 0.5% CCl4-treated HepG2 cells. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄⁄p < 0.01 and ⁄p < 0.05 as compared with control group, ##p < 0.01 and #p < 0.05 as compared with CCl4 group.
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Inflammation is an important pathological mechanism propagating CCl4-induced liver injury [35,36]. CCl4-treated mice showed severe inflammation owing to CCl4-induced ROS formation. COX-2 and iNOS are two major inflammatory mediators considered to be inflammation markers, which play pivotal roles in the development of certain inflammatory diseases [37,38]. Antioxidant is responsible for attenuation of inflammatory response in liver, suggesting a link between oxidative stress and inflammation [39]. In our experiment, pretreatment with NHDC suppressed hepatic damage (Fig. 2) and the expression of COX-2 and iNOS (Fig. 3). ROS-induced lipid peroxidation mediate pro-inflammatory effects via the activation of NF-jB [40]. Correspondingly, our results showed that suppression of inflammation by NHDC was mediated through the inhibition of NF-jB pathway and down-regulation of COX-2, iNOS and IL-6 (Fig. 9), which is in consistent with previous findings [41,42]. These results, for the first time, suggested the anti-inflammatory ability of NHDC in CCl4-induced liver injury. CCl4-induced hepatocyte apoptosis play a major role in the development of liver disease [43]. Caspase-3 is one of the key executioners of apoptosis, the activation of caspase-3 has been considered to be an indicator of apoptosis. Our Western blotting assay illustrated the elevation of cleaved caspase-3 in CCl4 group as compared to control group, however, its expression was inhibited with pretreatment of NHDC, which further supported previous studies [44]. Caspase-8 is one of the initiator caspases, normally associated with extrinsic apoptotic pathway [45]. In extrinsic apoptotic pathway, apoptotic signals are initiated by death receptors ligation with their cognate ligands, leading to the recruitment of FASassociated death domain protein and release caspase 8, which activates caspase 3 and initiates cell death ultimately [46]. In the current study, CCl4 increased caspase-8 expression in the liver of mice, which was markedly decreased by NHDC. This result may implied that CCl4-induced apoptosis via an extrinsic pathway [44]. In addition to the inhibitory effect of NHDC on CCl4-induced liver injury, as seen by an increase of antioxidant enzyme activities. Intracellular antioxidants are critical for the effective detoxification of free radicals. Previous studies have reported that CCl4 administration decreased antioxidant enzymes activities, including CAT, SOD and GP-X [47,48]. The loss of antioxidant enzyme activities is relevant to weaken of ability of the liver during CCl4 intoxication. Our results indicated that NHDC treatment significantly restored CAT, T-SOD and GP-X activities as well as GSH level, compared with CCl4 group (Fig. 4). These findings suggested that NHDC has considerable antioxidant activity and suppressed CCl4-induced oxidative liver injury. In in vitro study, HepG2 cell line was employed to confirm hepatoprotective activity of NHDC against CCl4-induced oxidative damage. HepG2 cell, a human-derived cell line, minimizes any species related differences. Cell viability directly reflects the extent of cellular stress-response. In our study, pretreatment with NHDC significantly prevented the loss of cell viability induced by CCl4 (Fig. 5). In addition, NHDC inhibited CCl4-induced intracellular ROS in a dose-dependent manner (Fig. 6). TBARS is an indicator of oxidative damage, as implicated in the pathogenesis of hepatic injury and cell membrane integrity [48]. In the present study, TBARS level was increased in CCl4-intoxicated cells. However, NHDC reduced TBARS level dose-dependently (Fig. 7). Consistently, CAT, T-SOD and GP-X activities and GSH level were restored by NHDC pretreatment in HepG2 cells (Fig. 8). Therefore, we propose that anti-oxidative effect of NHDC attenuate CCl4-induced cellular oxidative stress by scavenge free radical. In summary, our in vivo and in vitro studies demonstrated the effectiveness of NHDC in the attenuation of CCl4-induced liver injury, possibly by reducing oxidative stress, inflammation, and apoptotic cell death, which is in agreement with its antioxidant
activity. This study provides evidence that NHDC may be an alternative treatment for oxidative related liver diseases. Conflict of interest statement The authors declare that there is no financial conflict of interests. Acknowledgements This work was supported by the National Natural Science Foundation of China (NSFC-21005064, 21035005) the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (2011[508]) and the Fundamental Research Funds for the Central Universities (XDJK2010B008, XDJK2013B009). References [1] M.S. Shiao, J.J. Chiu, B.W. Chang, J. Wang, W.P. Jen, Y.J. Wu, Y.L. Chen, In search of antioxidants and anti-atherosclerotic agents from herbal medicines, BioFactors 34 (2) (2008) 147–157. [2] Y.Z. Zhu, S.H. Huang, B.K. Tan, J. Sun, M. Whiteman, Y.C. Zhu, Antioxidants in Chinese herbal medicines: a biochemical perspective, Nat. Prod. Rep. 21 (4) (2004) 478–489. [3] Y.T. Chen, R.L. Zheng, Z.J. Jia, Y. Ju, Flavonoids as superoxide scavengers and antioxidants, Free Radic. Biol. Med. 9 (1) (1990) 19–21. [4] J.W. Chen, Z.Q. Zhu, T.X. Hu, D.Y. Zhu, Structure–activity relationship of natural flavonoids in hydroxyl radical-scavenging effects, Acta Pharmacol. Sin. 23 (7) (2002) 667–672. [5] G. Sichel, C. Corsaro, M. Scalia, A.J. Di Bilio, R.P. Bonomo, In vitro scavenger activity of some flavonoids and melanins against O2-(), Free Radic. Biol. Med. 11 (1) (1991) 1–8. [6] D.H. Waalkens-Berendsen, M.E. Kuilman-Wahls, A. Bar, Embryotoxicity and teratogenicity study with neohesperidin dihydrochalcone in rats, Regul. Toxicol. Pharmacol. 40 (1) (2004) 74–79. [7] A. Frydman, O. Weisshaus, D.V. Huhman, L.W. Sumner, M. Bar-Peled, E. Lewinsohn, R. Fluhr, J. Gressel, Y. Eyal, Metabolic engineering of plant cells for biotransformation of hesperedin into neohesperidin, a substrate for production of the low-calorie sweetener and flavor enhancer NHDC, J. Agric. Food Chem. 53 (25) (2005) 9708–9712. [8] G.E. DuBois, G.A. Crosby, R.A. Stephenson, Dihydrochalcone sweeteners. A study of the atypical temporal phenomena, J. Med. Chem. 24 (4) (1981) 408– 428. [9] G.E. DuBois, G.A. Crosby, R.A. Stephenson, R.E. Wingard Jr., Dihydrochalcone sweeteners. Synthesis and sensory evaluation of sulfonate derivatives, J. Agric. Food Chem. 25 (4) (1977) 763–772. [10] R.P. Batzinger, S.Y. Ou, E. Bueding, Saccharin and other sweeteners: mutagenic properties, Science 198 (4320) (1977) 944–946. [11] J.P. Brown, P.S. Dietrich, Mutagenicity of plant flavonols in the Salmonella/ mammalian microsome test: activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources, Mutat. Res. 66 (3) (1979) 223–240. [12] J.T. MacGregor, L. Jurd, Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium, Mutat. Res. 54 (3) (1978) 297–309. [13] R.K. Sahu, R. Basu, A. Sharma, Genetic toxicological of some plant flavonoids by the micronucleus test, Mutat. Res. 89 (1) (1981) 69–74. [14] J.M. Choi, B.S. Yoon, S.K. Lee, J.K. Hwang, R. Ryang, Antioxidant properties of neohesperidin dihydrochalcone: inhibition of hypochlorous acid-induced DNA strand breakage, protein degradation, and cell death, Biol. Pharm. Bull. 30 (2) (2007) 324–330. [15] Y. Nakamura, S. Watanabe, N. Miyake, H. Kohno, T. Osawa, Dihydrochalcones: evaluation as novel radical scavenging antioxidants, J. Agric. Food Chem. 51 (11) (2003) 3309–3312. [16] J. Suarez, M.D. Herrera, E. Marhuenda, In vitro scavenger and antioxidant properties of hesperidin and neohesperidin dihydrochalcone, Phytomedicine 5 (6) (1998) 469–473. [17] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1–2) (1983) 55–63. [18] R.L. Heath, L. Packer, Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation, Arch. Biochem. Biophys. 125 (1) (1968) 189–198. [19] W.F. Beyer Jr., I. Fridovich, Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions, Anal. Biochem. 161 (2) (1987) 559–566. [20] H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121–126. [21] A. Wendel, Glutathione peroxidase, Methods Enzymol. 77 (1981) 325–333.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Protective effects of neohesperidin dihydrochalcone against carbon tetrachloride-induced oxidative damage in vivo and in vitro Lihua Hu a, Lingrui Li a, Demei Xu a, Xiaomin Xia a, Ruxian Pi b, Duo Xu a, Wenchao Wang a, Hong Du a, Erqun Song a, Yang Song a,⇑ a Key Laboratory of Luminescence and Real-Time Analytical Chemistry, Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, People’s Republic of China b Department of Hepatobiliry Surgery, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 6 November 2013 Received in revised form 15 January 2014 Accepted 5 February 2014 Available online 14 February 2014 Keywords: Hepatotoxicity Antioxidant Liver injury COX-2 iNOS HepG2 cell
a b s t r a c t The purpose of this study was to investigate the possible hepatoprotective effects of neohesperidin dihydrochalcone (NHDC) on carbon tetrachloride (CCl4)-induced acute oxidative injury in vivo and in vitro. In a mouse model, intraperitoneal injection of CCl4 resulted in a significant increase in serum aspartate transaminase (AST) and alanine transaminase (ALT) activities. Histopathological examination revealed severe hepatocyte necrosis and destruction of architecture in liver lesions, and immunohistochemical staining illustrated a remarkable enhancement of COX-2 and iNOS expression. The levels of hepatic antioxidant, such as, catalase (CAT), total superoxide dismutase (T-SOD), glutathione peroxidase (GP-X) and glutathione (GSH) were decreased, compared to the control group. However, pretreatment of NHDC for six consecutive days significantly ameliorated these changes. Moreover, Western blotting assay indicated pretreatment with NHDC also down-regulated CCl4-induced protein expressions of NF-jB, IL-6, caspase 3 and caspase 8. In HepG2 cell model, CCl4-treatment caused significant decrease in cell viability, antioxidant activities and GSH level, increase in intracellular reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) level. Interestingly, pretreatment of NHDC effectively relieved CCl4-induced oxidative damage in a dose-dependent manner. In conclusion, NHDC appeared to possess promising anti-oxidative and anti-inflammatory capacities, it is possible to be used as a hepatoprotective agent. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Free radicals and oxidative stress play pivotal roles in the pathogenesis of a number of human diseases, such as cardiovascular diseases and diabetes, which led to the enthusiastic use of antioxidants in the treatment and prevention of these diseases in clinic [1,2]. In general, flavonoids possess high antioxidant Abbreviations: ALT, alanine transaminase; AST, aspartate transaminase; CAT, catalase; CCl4, carbon tetrachloride; COX-2, cyclooxygenase-2; DAB, 3,30 -diaminobenzidine tetrahydrochloride; DCFH-DA, 20 ,70 -dichlorodihydrofluorescein diacetate; GP-X, glutathione peroxidase; GSH, glutathione; HepG2, human hepatoma cell line; IL-6, interleukin 6; iNOS, inducible nitric oxide synthase; NF-jB, nuclear factor-kappa B; NHDC, neohesperidin dihydrochalcone; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; T-SOD, total superoxide dismutase. ⇑ Corresponding author. Present address: College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing 400715, People’s Republic of China. Tel.: +86 23 68251503; fax: +86 23 68251225. E-mail addresses: [email protected], [email protected] (Y. Song). http://dx.doi.org/10.1016/j.cbi.2014.02.003 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
activities directed toward O 2 , HO and alkoxyl radicals [3–5]. Neohesperidin dihydrochalcone (NHDC, 3,5-dihydroxy-4-(3-hydroxy-4-methoxyhydrocinnamoyl)phenyl 2-O-b-L-rhamnopyranosyl-b-D-glucopyranoside, structures presented in Fig. 1) belongs to a family of bycyclic flavonoids dihydrochalcones, which have two benzenoid rings joined by a three-carbon bridge. NHDC is manufactured by catalytic hydrogenation of neohesperidin in industry [6]. Essentially, NHDC is the ring-opening product of neohesperidin. NHDC is approximately 1500 times sweeter than sucrose [7], which has been used in food, beverage and pharmaceutical as an intense, low caloric artificial sweetener for half a century [8,9]. NHDC is well tolerated and is not associated with any particular toxicity as a well-known sweetener. No adverse effects were observed at NHDC levels of up to 5% of the diet of Wistar rats in embryotoxicity and teratogenicity studies [6]. The mutagenicity of NHDC was assessed in different experimental models, and no mutagenic activity was detected [10–13]. These results encouraged
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Three hours after final NHDC treatment, mice were injected with CCl4 (10 mL/kg body weight, v/v = 1:49 in olive oil). Twenty-four hours after the last dose of injection, mice were anesthetized with CO2. Blood samples were collected by cardiac puncture and allowed to clot for 45 min at room temperature. The livers were excised careful, washed twice with saline, blotted dry on a filter paper, weighed, then cut into two pieces. One halves were used for histopathological and immunohistochemical analysis. Other halves were used for hepatic homogenate preparation. Fig. 1. Chemical structures of NHDC.
us to investigate the antioxidant activity of NHDC. In recent studies, NHDC showed outstanding radical scavenging activity against ABTS+, O 2 , HO , H2O2 and HOCl [14,15]. In another study, NHDC is more effective in the inhibition of HOCl-induced protein degradation, plasmid DNA strand cleavage and cell death than ascorbic acid, mannitol and butylated hydroxy toluene [16]. However, to the best of our knowledge, the antioxidant activity of NHDC towards CCl4-induced acute hepatic injury has never been explored in vivo or in vitro. In the present study, mice and HepG2 cell models were employed to investigate the protective effects and mechanism of NHDC on carbon tetrachloride (CCl4)-induced acute liver injury.
2.3. Biochemical parameters of liver function Serums were separated by centrifugation of blood at 600g for 15 min. Serum AST and ALT activities were measured by diagnostic kits (Nanjing Jiancheng Institute of Biotechnology). 2.4. Histopathological examination Halves of liver were fixed for 24 h in 10% buffered formalin. They were embedded in paraffin and sectioned for 5 lm thickness. After hematoxylin–eosin (H&E) staining, slides were observed for histopathological changes using Nikon TE2000 fluorescence microscope (Nikon, Japan). Representative images were presented.
2. Materials and methods
2.5. Immunohistochemical staining
2.1. Materials
The paraffin-embedded sections were deparaffinized and rehydrated. Antigen retrieval was performed with a 1 mM EDTA buffer (pH = 9.0) in a microwave for 3 min. Then, the following steps were performed according to the instructions of Histostain™-plus and DAB substrate kits. Briefly, 3% H2O2 was used to block endogenous peroxidase activity for 20 min and non-specific protein binding was blocked by normal goat serum for 20 min. The treated slides were incubated in a moist box at 4 °C overnight with rabbit iNOS and COX-2 antibodies (1:100, dilution) followed by incubation biotin-labeled goat anti-rabbit IgG and horseradish peroxidaseconjugated streptavidin for 50 min, respectively. DAB fluid including 1 mL distilled water, 50 lL H2O2 (20) and 50 lL DAB (20) was used in color development and counterstained by hematoxylin. Images were taken by a light microscopy (magnification, 100, Nikon Eclipse Ti-SR).
NHDC (CAS number, 20702-77-6), CCl4, 20 ,70 -dichlorodihydrofluorescein diacetate (DCFH-DA) and olive oil were purchased from Aladdin Reagent Database Inc. (Chengdu, China). Diagnostic kits used for the determination of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GP-X), aspartate transaminase (AST), alanine transaminase (ALT), reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were obtained from the Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Histostain™-plus kit and 3,30 -diaminobenzidine tetrahydrochloride (DAB) substrate kit were provided by Zhongshan Golden Bridge Biotechnology (Beijing, China). Inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and b-actin antibodies were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Interleukin-6 (IL-6), nuclear factor-kappa B (NF-jB), caspase 3 and caspase 8 antibodies were purchased from Proteintech Group Inc. (Wuhan, China). All other chemicals used were of highest commercial grade. 2.2. Animals, treatment and tissue collection The animal experiments were performed in accordance with the guideline of the Animal Care Committee of Southwest University. Male Kunming mice (22 ± 2 g) were purchased from Chongqing Tengxin Biotechnology Co. Animals were maintained under standard conditions of humidity (50%), temperature (25 ± 2 °C) in a 12 h light and 12 h dark cycle. They were fed standard rodent chow and had free access to water, acclimatized for at least one week prior to use. Mice were assigned to four groups randomly with 6 mice in each group: (1) control group, administered appropriate vehicles throughout; (2) NHDC group, received at a daily dose of NHDC (100 mg/kg body weight, dissolved in a 8% Tween 80/H2O vehicle and prepared before use) by oral gavage for 6 consecutive days; (3) CCl4 group, received saline once daily for 6 consecutive days. Three hours after final saline treatment, mice were intraperitoneally injected with CCl4 (10 mL/kg body weight, v/v = 1:49 in olive oil); (4) NHDC + CCl4 group, received at a daily dose of NHDC (100 mg/kg body weight) by oral gavage for 6 consecutive days.
2.6. Hepatic homogenate preparation Half livers were rinsed in ice-cold physiological saline and homogenized in Tris–HCl buffer (0.01 M, pH = 7.4) to give a 10% homogenates. Homogenates were centrifuged at 3000 rpm, 4 °C for 10 min and supernatants were collected for catalase (CAT), total superoxide dismutase (T-SOD) and glutathione peroxidase (GP-X) activities and reduced glutathione (GSH) level measurement. 2.7. Cell culture and treatment The human hepatoma cell line (HepG2) was obtained from Third Military Medical University, Chongqing, China. Cells were grown on RPMI 1640 medium with 10% fetal bovine serum (HyClone, USA), antibiotics (100 U/mL penicillin, 100 lg/mL streptomycin) at 37 °C and 5% CO2. Exponential growth phase cells were transferred onto 96-well culture plates at 1 104 cells/well and permitted to adhere overnight at 37 °C. Cells were treated with NHDC (10, 20 or 30 lM) or vehicle for 1 h, then, medium was replaced with fresh medium containing 0.5% (v/v) CCl4 for 24 h. Following incubation, cells were lysed with 300 lL of 0.1% Triton X-100 on ice for 10 min and sonicated for 10 s. Homogenates were centrifuged at 12,000 rpm, 4 °C for 20 min, and supernatants were collected for further investigation.
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2.8. Cell viability assay
by measuring the concentration of red adduct (absorbance at 532 nm) according to the methods of Heath and Parker [18].
Cell viability was evaluated using the MTT assay [17]. After incubation with CCl4 for 3 h, cells were treated with MTT (final concentration of 5 mg/mL) for 4 h at 37 °C. The MTT-containing medium was removed and 100 lL of DMSO were added to all wells, mixed thoroughly with a 10 min shake. The optical density (OD) of each well was measured at 490 nm using a microplate reader (BioTek ELX800). 2.9. Determination of reactive oxygen species (ROS) generation ROS level in HepG2 cells was determined using DCFH-DA as fluorescent probe. Fluorescence was recorded with an excitation wavelength of 488 nm and an emission wavelength of 526 nm (F7000, HITACHI). The fluorescence images were captured with a fluorescence microscope (OLYMPUS IX71) in order to obtain comparable data. 2.10. Determination of lipid peroxidation The level of TBARS was measured by commercial kits according to the manufacturer’s instruction (Nanjing Jiancheng Institute of Biotechnology). In brief, the formation of TBARS was determined
Table 1 Effect of NHDC on serum biochemical parameters in 0.5% CCl4-intoxicated mice. Data represent the mean ± SEM of three independent experiments. Significant differences are indicated by ⁄⁄⁄p < 0.001 as compared with control group, ##p < 0.01 and #p < 0.05 as compared with CCl4 group. Groups
Treatment
AST (IU/L)
ALT (IU/L)
Group Group Group Group
Control NHDC CCl4 NHDC + CCl4
54.8 ± 20.76 32.84 ± 15.79 184.44 ± 36.48⁄⁄ 56.36 ± 32.85##
17.52 ± 1.97 18.54 ± 1.83 329.92 ± 3.49⁄⁄⁄ 315.53 ± 7.05#
I II III IV
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2.11. Determination of antioxidants Levels of T-SOD, CAT, GP-X and GSH in HepG2 cells (or liver homogenates) were measured by commercial kits according to the manufacturer’s instructions (Nanjing Jiancheng Institute of Biotechnology). T-SOD activity was determined based on the reaction of SOD and nitrotetrazolium blue chloride, the product was measured with spectrophotometer at its maximum absorbance of 550 nm [19]. CAT activity was determined by measuring the H2O2 decomposition (absorbance at 240 nm) [20]. GP-X activity was measured by following the absorbance at 412 nm based on the oxidation of NADPH with H2O2 [21]. GSH level was measured by using the method of Anderson, the colorimetric reaction was monitored at 405 nm spectrophotometrically as 5,50 -dithiobis-2nitrobenzoic acid (DTNB) was reduced into 2-nitro-5-thiobenzoic acid (TNB) by GSH [22].
2.12. Western blotting Briefly, proteins were extracted from liver, separated by electrophoresis in 10% SDS–PAGE and transferred onto nitrocellulose membrane. After blocking with 10% skimmed milk, membranes were incubated with primary antibodies of rabbit polyclonal antibodies IL-6, NF-jB, caspase 3 and caspase 8 at room temperature for 4 h. After washing, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). Finally, immune-reactive protein bands were detected by the HRP substrate DAB system. Representative blots were chosen from three independent experiments. Densitometric analysis of western blots was performed with the use of ImageJ software. Protein levels were standardized by comparison with b-actin.
Fig. 2. Effect of NHDC on liver histopathology changes stained with hematoxylin–eosin. (A) Control mice, (B) mice treated by NHDC at 100 mg/kg, (C) CCl4-intoxicated mice and (D) mice treated by NHDC at 100 mg/kg and CCl4. Typical examples were shown from three independent experiments. Original magnification: 400x. The arrows show necrotic area.
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2.13. Statistical analysis
3. Results
All experiments were performed with three times independently. Data were presented as means ± SEM. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by least significance difference (LSD) multiple comparison tests using SPSS 18.0 software. p < 0.05 was considered to be significant.
3.1. Effect of NHDC on serum AST and ALT activities Serum AST and ALT are two common biomarkers of liver damage. The effect of NHDC on AST and ALT activities in serum were shown in Table 1. In the control group, AST and ALT activities were 54.8 ± 20.76 and 17.52 ± 1.97 IU/L, respectively. Pretreatment with
Fig. 3. Effect of NHDC on hepatic (A) COX-2 and (B) iNOS expression in CCl4-intoxicated mice. Typical examples were shown from three independent experiments. Original magnification: 100. The arrows show positive staining.
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NHDC alone has no significant effect on AST and ALT activities. However, Serum AST and ALT activities were significantly (p < 0.001) elevated after CCl4 injection, as compared to the control group. Surprisingly, pretreatment with NHDC at dose of 100 mg/ kg/day significantly decreased AST and ALT activities as compared to CCl4 group, p < 0.01 and p < 0.05, respectively. 3.2. Effect of NHDC on CCl4-induced hepatic necrosis In Fig. 2, the effect of NHDC on CCl4-induced hepatic histopathological damage was presented. Intraperitoneal injection of CCl4 resulted in histological changes in liver morphology, including massive cell necrosis and loss of hepatocyte architecture around the blood vessels (Fig. 2C). However, NHDC extensively alleviated CCl4-induced hepatic histopathological damage (Fig. 2D).
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in CCl4 group, compared to the control group (p < 0.05) (Fig. 4D). However, pretreatment with NHDC significantly elevated GSH levels in liver tissues compared to CCl4 group (p < 0.05).
3.5. Effect of NHDC on HepG2 cell viability The protective role of NHDC was further evaluated in human hepatocellular carcinoma HepG2 cells. In Fig. 5, MTT results showed that CCl4 caused significant loss of cell viability (about 20%), compare to the control group, p < 0.01. Interestingly, preincubation of NHDC in CCl4-intoxicated HepG2 cells increased cell viability in a dose-dependent manner, significant difference was found in the group of 30 lM NHDC (p < 0.05 compared to CCl4 group).
3.3. Effect of NHDC on CCl4-induced inflammatory response COX-2 and iNOS are two major inflammatory mediators implicated in inflammation. Immunohistochemical analysis revealed considerable up-regulation of hepatic COX-2 and iNOS proteins after CCl4 injection (shown as brown-colored stain), whereas the levels of COX-2 and iNOS were low in the control group (Fig. 3). However, NHDC attenuated the expression of proteins COX-2 and iNOS considerably. 3.4. Effect of NHDC on hepatic antioxidants in mice CCl4-intoxication resulted in a significant decrease in hepatic CAT, T-SOD and GP-X activities compared to the control group, p < 0.05 (Fig. 4A–C). On the contrary, pretreatment with NHDC restored these hepatic antioxidant enzyme activities. GSH, a sensitive marker of oxidative stress, was detected to further evaluate the degree of oxidative injury. Hepatic GSH level significantly decreased
Fig. 5. Effect of NHDC on 0.5% CCl4-treated HepG2 cell viability. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄p < 0.05 as compared to control group, #p < 0.05 as compared to CCl4 group.
Fig. 4. Effect of NHDC on hepatic (A) T-SOD, (B) CAT and (C) GP-X activities and (D) GSH level in CCl4-intoxicated mice. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄⁄⁄p < 0.001, ⁄⁄p < 0.01 and ⁄p < 0.05 as compared with control group, ###p < 0.001, ##p < 0.01 and #p < 0.05 as compared to CCl4 group.
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3.6. Effect of NHDC on ROS generation in HepG2 cells Intracellular ROS production plays an important role in CCl4induced oxidative damage. ROS level was investigated using DCFH-DA as a fluorescence probe. We observed that CCl4 intoxication caused a significant increase in ROS production (p < 0.05) (Fig. 6A). However, ROS level was inhibited by NHDC significantly in the 30 lM NHDC group (p < 0.05), compared to CCl4 group. Furthermore, by applying fluorescence microscopy, it was demonstrated that ROS level was considerably elevated in HepG2 cells by the treatment of CCl4 compared to unstimulated cells. Consistently, ROS was decreased by the pretreatment of NHDC in dose-dependent manner (Fig. 6B).
3.7. Effect of NHDC on lipid peroxidation in HepG2 cells Thiobarbituric acid reactive substances (TBARS) are formed as a byproduct of lipid peroxidation. After treatment with CCl4, TBARS level increased by about 2-fold (Fig. 7). However, pretreatment with NHDC inhibited TBARS in HepG2 cells dose-dependently, 20 and 30 lM NHDC groups showed statistical significance, p < 0.01 against CCl4 group.
Fig. 7. Effect of NHDC on TBARS level in 0.5% CCl4-treated HepG2 cells. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄⁄p < 0.01 as compared with control group, ##p < 0.01 as compared with CCl4 group.
3.8. Effect of NHDC on antioxidants in HepG2 cells Intracellular levels of enzymatic and non-enzymatic antioxidants were shown in Fig. 8. CCl4-intoxicated HepG2 cells showed
Fig. 6. Effect of NHDC against 0.5% CCl4-induced ROS formation in HepG2 cells. (A) ROS level was presented by the fluorescent product of DCFH-DA. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄p < 0.05 as compared with control group, #p < 0.05 as compared with CCl4 group. (B) Cells were stained with DCFH-DA probe and imaged by fluorescence microscope.
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a significant decrease in CAT, T-SOD and GP-X activities and GSH content (p < 0.05 or p < 0.01 against the control group). Pretreatment with NHDC elevated these antioxidants dose-dependently. 3.9. Effect of NHDC on the protein expression in liver tissue Western blot analysis showed that the levels of IL-6, NF-jB, caspase 3 (cleaved form) and caspase 8 proteins were observably up-regulated in CCl4 group, which was subsequently reduced under treatment with NHDC (Fig. 9). 4. Discussion Flavonoids are ubiquitous polyphenolic compounds existing in plants, categorized into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones [23]. It is well known that one of the benefits of flavonoids is protection against cardiovascular disease and oxidative stress as antioxidants. [24]. Besides direct scavenging of free radicals, flavonoids can prevent tissue injury by activation of antioxidant enzymes [25], chelating of metal ion [26], reduction of a-tocopheryl radicals [27], inhibition of oxidases [28], mitigation of nitric oxide [29], increasing of uric acid levels [30], etc. As a group of minor flavonoids, dihydrochalcones have not been widely investigated for their strength of antioxidant activity. Interestingly, Dziedzic et al. demonstrated that dihydrochalcones have higher antioxidant capacity than corresponding chalcones and flavanones [31]. CCl4 causes severe liver damage with necrotic and apoptotic hepatocellular injury. In the principle of liver injury, oxidative stress resulting from CCl4 metabolism plays an important role. CCl4 was metabolized into trichloromethyl radical (CCl3 ) by the cytochrome p450, then reacts with oxygen to form trichloromethylperoxy
Fig. 9. Effects of NHDC on the protein expressions in CCl4-intoxicated mice. Protein expressions of IL-6, NF-jB, caspase 3 and caspase 8 were assessed by Western blotting. The particular procedure was performed as described in Material and Methods section and protein expressions were analyzed by SDS–PAGE electrophoresis. The house keeping gene, b-actin, was used as control. One of three representative experiments is shown.
radical (CCl3OO), resulting in protein and DNA damage and lipid peroxidation [32]. Therefore, diminishing free radical directly and retarding the propagation of oxidative chain reaction are practicable strategies to preventing CCl4-induced hepatotoxicity. CCl4-induced hepatic injury is a widely used experimental model in screening of hepatoprotective drugs [33]. The present study showed that NHDC possesses a hepatoprotective effect against CCl4-induced damage as evidenced by significant histopathological alterations, restoring antioxidant enzyme activities and reducing inflammation and apoptosis compared to CCl4treated mice in the absence of NHDC. According to previous study, CCl4-treatment altered membrane integrity and increased serum AST and ALT activities [34]. It is well-accepted that antioxidants reducing serum AST and ALT activities exert hepatoprotective activity, our results suggested a promising potential of NHDC on preventing CCl4-induced hepatotoxicity, Table 1.
Fig. 8. Effect of NHDC on (A) total SOD (T-SOD), (B) CAT and (C) GP-X activities and (D) GSH level in 0.5% CCl4-treated HepG2 cells. Data represent the mean ± SEM of three independent experiments. Significant differences were indicated by ⁄⁄p < 0.01 and ⁄p < 0.05 as compared with control group, ##p < 0.01 and #p < 0.05 as compared with CCl4 group.
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Inflammation is an important pathological mechanism propagating CCl4-induced liver injury [35,36]. CCl4-treated mice showed severe inflammation owing to CCl4-induced ROS formation. COX-2 and iNOS are two major inflammatory mediators considered to be inflammation markers, which play pivotal roles in the development of certain inflammatory diseases [37,38]. Antioxidant is responsible for attenuation of inflammatory response in liver, suggesting a link between oxidative stress and inflammation [39]. In our experiment, pretreatment with NHDC suppressed hepatic damage (Fig. 2) and the expression of COX-2 and iNOS (Fig. 3). ROS-induced lipid peroxidation mediate pro-inflammatory effects via the activation of NF-jB [40]. Correspondingly, our results showed that suppression of inflammation by NHDC was mediated through the inhibition of NF-jB pathway and down-regulation of COX-2, iNOS and IL-6 (Fig. 9), which is in consistent with previous findings [41,42]. These results, for the first time, suggested the anti-inflammatory ability of NHDC in CCl4-induced liver injury. CCl4-induced hepatocyte apoptosis play a major role in the development of liver disease [43]. Caspase-3 is one of the key executioners of apoptosis, the activation of caspase-3 has been considered to be an indicator of apoptosis. Our Western blotting assay illustrated the elevation of cleaved caspase-3 in CCl4 group as compared to control group, however, its expression was inhibited with pretreatment of NHDC, which further supported previous studies [44]. Caspase-8 is one of the initiator caspases, normally associated with extrinsic apoptotic pathway [45]. In extrinsic apoptotic pathway, apoptotic signals are initiated by death receptors ligation with their cognate ligands, leading to the recruitment of FASassociated death domain protein and release caspase 8, which activates caspase 3 and initiates cell death ultimately [46]. In the current study, CCl4 increased caspase-8 expression in the liver of mice, which was markedly decreased by NHDC. This result may implied that CCl4-induced apoptosis via an extrinsic pathway [44]. In addition to the inhibitory effect of NHDC on CCl4-induced liver injury, as seen by an increase of antioxidant enzyme activities. Intracellular antioxidants are critical for the effective detoxification of free radicals. Previous studies have reported that CCl4 administration decreased antioxidant enzymes activities, including CAT, SOD and GP-X [47,48]. The loss of antioxidant enzyme activities is relevant to weaken of ability of the liver during CCl4 intoxication. Our results indicated that NHDC treatment significantly restored CAT, T-SOD and GP-X activities as well as GSH level, compared with CCl4 group (Fig. 4). These findings suggested that NHDC has considerable antioxidant activity and suppressed CCl4-induced oxidative liver injury. In in vitro study, HepG2 cell line was employed to confirm hepatoprotective activity of NHDC against CCl4-induced oxidative damage. HepG2 cell, a human-derived cell line, minimizes any species related differences. Cell viability directly reflects the extent of cellular stress-response. In our study, pretreatment with NHDC significantly prevented the loss of cell viability induced by CCl4 (Fig. 5). In addition, NHDC inhibited CCl4-induced intracellular ROS in a dose-dependent manner (Fig. 6). TBARS is an indicator of oxidative damage, as implicated in the pathogenesis of hepatic injury and cell membrane integrity [48]. In the present study, TBARS level was increased in CCl4-intoxicated cells. However, NHDC reduced TBARS level dose-dependently (Fig. 7). Consistently, CAT, T-SOD and GP-X activities and GSH level were restored by NHDC pretreatment in HepG2 cells (Fig. 8). Therefore, we propose that anti-oxidative effect of NHDC attenuate CCl4-induced cellular oxidative stress by scavenge free radical. In summary, our in vivo and in vitro studies demonstrated the effectiveness of NHDC in the attenuation of CCl4-induced liver injury, possibly by reducing oxidative stress, inflammation, and apoptotic cell death, which is in agreement with its antioxidant
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