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fibrogenic responses in livers of thioacetamide-treated rats
journal of functional foods 14 (2015) 154–162
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Ameliorative effects of D-glucuronolactone on oxidative stress and inflammatory/fibrogenic responses in livers of thioacetamide-treated rats Po-Ju Chen a, Chih-Hsien Chiu a, Jung-Kai Tseng b, Kou-Tai Yang a, Yi-Chen Chen a,* a b
Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan School of Optometry, Chung Shan Medical University, Taichung 402, Taiwan
A R T I C L E
I N F O
A B S T R A C T
Article history:
D-Glucuronolactone (C6H8O6, lactone), naturally found in plant gums, is commercially ac-
Received 7 August 2014
claimed for its hepatoprotective effects. This study was to investigate whether lactone can
Received in revised form 14 January
attenuate thioacetamide (TAA)-induced liver fibrosis in a rat model. Results showed that
2015
lactone supplementation (75 mg kg−1 bw glucuronolactone) alleviated AST values in TAA-
Accepted 21 January 2015
intraperitoneally-injected rats (100 mg kg−1 bw TAA) and increased antioxidant capacity of
Available online
liver via elevations of antioxidant enzymes activities [superoxide dismutase (SOD) and glutathione peroxidase (GPx)], glutathione (GSH) and trolox equivalent antioxidative capacity
Keywords:
(TEAC) levels (p < 0.05). Down-regulated (p < 0.05) expression of inflammation including
Anti-inflammation
interleukin-6 (IL-6), nuclear factor-kappa B (NF-κB), activator protein 1 (AP-1), krüppel-like
Antioxidant
factor 6 (KLF-6), and fibrosis related fibrotic factors, i.e., alpha smooth muscle actin (α-SMA),
D-glucuronolactone
and collagen alpha1 (I) (COLα1) through lactone supplementation underlay the lower col-
Liver fibrosis
lagen contents and less severe liver damage on histopathology observations. Therefore,
Thioacetamide
hepatoprotection of lactone against TAA-induced liver fibrosis can be attributed to the amelioration of oxidative stress and inflammation. © 2015 Elsevier Ltd. All rights reserved.
1.
Introduction
In recent years, there has been a gradual rise in the number of people suffering from liver diseases, mainly caused by alcohol, toxins, drugs, viruses, and abnormal dietary habits. In Taiwan, chronic liver disease and cirrhosis ranked sixth among leading causes of death in 2013 (Ministry of Health and Welfare, Executive Yuan, Taiwan, 2014). Liver fibrosis is often related to escalating reactive oxygen species (ROS) in the body. Excessive ROS results in an inflammation of hepatocytes and cytokine
secretion, and activates hepatic stellate cells (HSCs) into myofibroblast-like cells with secreting extracellular matrix (ECM) proteins at the site of tissue repairs, further leading to hepatic fibrosis (Kim, Kim, Kim, Lee, & Lee, 2011). The characteristic of hepatic fibrosis features a progressive accumulation of ECM proteins, produced by activated HSCs. It has been indicated that HSCs are associated with inflammation-related genes (e.g., IL1β, IL6, and TNF-α) (Bataller & Brenner, 2005) and activated by many transcriptional factors like nuclear factor-kappa B (NFκB), activator protein 1 (AP-1), and krüppel-like factor 6 (KLF6), as well as alpha smooth muscle actin (α-SMA) and collagen
* Corresponding author. Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan. Tel.: +886 2 33664180; fax: +886 2 27324070. E-mail address: [email protected] (Y.-C. Chen). http://dx.doi.org/10.1016/j.jff.2015.01.026 1756-4646/© 2015 Elsevier Ltd. All rights reserved.
journal of functional foods 14 (2015) 154–162
alpha 1 (COLα1) (Chen et al., 2011; Difeo, Martignetti, & Narla, 2009; Mann & Smart, 2002). Based on potential reversibility of hepatic fibrosis, researchers have paid much attention to antifibrotic studies. Thioacetamide (CH3C(S)NH2, TAA) was found to cause liver damage, and hence, used in a number of research on liverrelated studies as a hepatotoxicant to induce acute and chronic liver diseases due to several advantages as follows: 1) high specificity for liver and regiospecificity for perivenous area; 2) relatively short half-life for a rapid induction of acute liver damage; 3) large window of time between its necrogenic effects and liver failure for better observation (Mehendale, 2005). TAA was found to be toxic after being metabolized (Chieli & Malvaldi, 1984). TAA is metabolically converted to thioacetamide-Soxide (TASO), acetamide and sulfate through microsomal mixedfunction oxidase system. TASO leads to centrilobular hepatic necrosis. A further metabolism causes biotransformation of TASO to thioacetamide-S, S-dioxide (TASO2), covalently binding to proteins with forming acetylimidolysine derivatives which act as hepatotoxic compounds. Originally, D-glucuronolactone (C6H8O6, lactone), also called D-glucuronic acid gamma-lactone, is a natural metabolite of glucose in animal livers and also found in plant gums with a complex form (Finnegan, 2003). It is commercially produced by nitric acid oxidation of starch and has been widely used as a common ingredient in energy drinks and also commercially acclaimed for its function of providing energy (Scientific Opinion of the Panel on Food Additives and Nutrient Sources Added to Food, 2009). Despite using lactone as a food ingredient, the safety of lactone has been debated. Ahrens, Douglass, Flynn, and Ward (1987) also indicated that there are no significant influences on growth performance of rats receiving the lactone. Scientific Opinion of the Panel on Food Additives and Nutrient Sources Added to Food (2009) in European Food Safety Authority reported that Scientific Committees on Food has examined a long-term toxicity of the lactone on both rat and hamster models. There are some conclusions below: 1) No significant treatment-related side effects on animals except kidney at high doses (600 and 1000 mg kg−1 bw day−1); 2) No-observedadverse-effect level (NOAEL) of the lactone in this experiment was 1000 mg kg−1 bw day−1. Hence, lactone supplementation seems to be safe at least with controllable dose. Lactone, in mammals, can be hydrolyzed to D-glucuronic acid, and converted into L-gulonolactone reversibly, as well as D-glucaric acid (Marsh, 1963). It has been indicated that D-glucuronic acid plays a crucial role in Phase II reaction of drug metabolism by conjugating with xenobiotics and other foreign compounds to form more hydrophilic substances, which are easily excreted in the urine (Hanausek, Walaszek, & Slaga, 2003). In addition, D-glucaric acid is normally observed in mammalian urine without toxicity, and its derivatives are also demonstrated to prevent breast, prostate, and colon cancer (Walaszek et al., 1997). However, few studies on ameliorative effects or the molecular mechanism of lactone on liver fibrosis have been available until now. Therefore, the current study was to induce a liver fibrosis of rats by an injection of TAA. Then, the ameliorative effects of lactone on TAA-induced liver fibrosis were investigated as the following aspects: 1) antioxidative capacities, 2) anti-inflammation, and 3) anti-fibrosis.
2.
Materials and methods
2.1.
Animals and treatments
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The animal use and protocol were reviewed and approved by National Taiwan University Animal Care Committee (IACUU No.: 100-15). Thirty-six male Wistar rats of 5 weeks in age were purchased from BioLASCO Taiwan Co., Ltd (Taipei, Taiwan), and acclimatized for one week before commencement of the experiment. Two rats were housed in each cage in an animal room at 22 ± 2 °C with a 12/12 h light–dark cycle, and randomly assigned to one of the following groups (n = 12 per group): 1) Control group: sterilized saline (intraperitoneally injection, i.p.) + 0.5 mL normal distilled water (oral gavage); 2) TAA group: 100 mg kg−1 bw TAA (i.p.) + 0.5 mL normal distilled water (oral gavage); 3) Lactone group: 100 mg kg−1 bw TAA (i.p.) + 75 mg lactone kg−1 bw in 0.5 mL normal distilled water (oral gavage). Animal experimental period lasted for 8 weeks with injections of TAA three times weekly (Monday, Wednesday, and Friday) and oral gavage daily. Chow diets containing 487 g carbohydrate, 239 g protein, 50 g fat, 51 g fiber, and 70 g ash kg−1 (Laboratory Rodent Diet 5001, PMI Nutrition International/ Purina Mills LLC, Richmond, IN, USA) with water were provided ad libitum. Lactone was kindly offered from Forever Chemical Co., Ltd. (Tauyan County, Taiwan), and TAA was purchased from Sigma Co. (St. Louis, MO, USA). Average daily food and water intakes per cage were calculated as volumes of food and water intakes on per rat daily basis, respectively. All rats were fasted overnight before euthanized by CO2 in the final day of experiment. Liver, heart, and kidney from each rat were removed, weighted and then stored at −80 °C for further analyses.
2.2.
Determination of serum liver damage indices
Blood samples were collected via abdominal aorta, and centrifuged at 3000 × g for 10 min to obtain sera, stored at −80 °C. Liver damage indices [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] were analyzed using commercial enzymatic kits with an ESPOTCHEMTMEZ SP4430 biochemistry analyzer (ARKRAY Inc., Kyoto, Japan).
2.3.
Preparation of liver homogenate
A 0.5 g sample of liver was homogenized on ice with 4.5 mL phosphate buffer saline (PBS, pH 7.0, containing 0.25 M sucrose). After a centrifugation (12,000 × g for 30 min) at 4 °C, supernatant was collected for further analyses. Protein levels in supernatant were measured by a Bio-Rad protein assay kit (Cat. #: 500-0006, Bio-Rad Laboratories, Inc., Hercules, CA, USA) using bovine serum albumin as a standard.
2.4.
Determination of antioxidant capacity in livers
The liver thiobarbituric-acid-reactive-substances (TBARS), glutathione (GSH), trolox equivalent antioxidative capacity (TEAC) levels, as well as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were determined to represent hepatic antioxidant capacities. Lipid peroxidation (TBARS) was determined by adding TBA solution
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and trichloroacetic acid–HCl (TCA–HCl) solution to homogenate. Mixture was reacted at 95 °C for 30 min, followed by centrifugation at 4000 × g for 15 min and then read at 535 nm. For measuring GSH contents, homogenates were reacted with TCA and centrifuged at 9000 × g for 5 min to obtain supernatant. Supernatant reacted with 2,2-dithio-bis-nitrobenzoic acid (DTNB) for 5 min, and then read at 412 nm.The TBARS and GSH values were calculated by taking the extinction coefficients of MDA to be 1.56 × 105 M−1 cm−1 at 532 nm and 2-nitro-5-thiobenzoic acid to be 1.36 × 104 M−1 cm−1 at 412 nm, respectively. In the assay of TEAC, homogenates were mixed with ABTS, peroxidase, as well as H2O2 under a dark room, and then measured at 734 nm after 10 min. The decrease in absorption at 734 nm was used to calculate the TEAC value. A standard curve was plotted for trolox on scavenging ABTS+ capacity and was used to calculated as the TEAC. For the purpose of determining SOD, homogenate added with Tris–HCl buffer (50 mM, pH 8.2) was prepared in advance and then the mixture was reacted with pyrogallol (0.2 mM) followed by measurement of the absorbance at 420 nm. One unit of SOD activity was defined as the amount of enzyme that inhibited the autoxidation of pyrogallol by 50%. CAT activity was measured by mixing homogenate with H2O2, and calculated by taking the extinction coefficient of H2O2 to be 39.5 M−1 cm−1 at 240 nm. One unit of CAT was expressed as the amount of enzyme that decomposes 1 mole H2O2 per minute at 25 °C. The measurement of GPx activity was briefly described as following. Homogenate was mixed with a reaction solution containing 100 mM PBS (pH 7.0), 10 mM EDTA, 10 mM NaN3, 2 mM NAPDH, 10 mM GSH, and 1 U/mL GSH reductase. After 5 min, H2O2 (2.5 mM) was then added and measured at 340 nm in a 3-min interval. Hepatic GPx activity was calculated by taking the extinction coefficient of NADPH to be 6.22 × 106 nM−1 cm−1. The liver TBARS, GSH, and TEAC levels were demonstrated as nmole MDA equivalent mg−1 protein, nmole mg−1 protein, and nmole mg−1 protein, respectively. The liver SOD, CAT, and GPx were demonstrated as unit mg−1 protein, unit mg−1 protein, and nmole NADPH oxidized min−1 mg−1 protein, respectively.
2.5.
Determination the level of TGF-β and IL-1β in livers
The hepatic TNF-α and IL-1β levels were measured by using enzyme-linked immunosorbent assay (ELISA) and were conducted according to the commercial manufacturer’s instructions (TNF-α and IL-1β kits, eBioscience Inc., San Diego, CA, USA). An aliquot of liver containing 100–200 ng of protein was extracted in the liver homogenate. First, the capture antibody (antiTNF-α and anti-IL-1β) was diluted with a coating buffer as a working solution. Each well of flat-bottom 96-well ELISA plates was coated with working solution and then incubated at 4 °C overnight. Thereafter, the plates were rinsed three times with washing buffer (1X PBS with 0.05% Tween-20). Then, the reagent diluent were added into plates and reacted for 1 h and rinsed as foregoing way. After prepared samples were loaded and incubated for 2 h, the plates were rinsed in the same way as described previously, followed by adding assay diluent reagent, incubation for 2 h and rinsed again. The working dilution of streptavidin–HRP was added to each well and then incubated for 20 min. Finally, the mixture was added with substrate solution. After a 30-min incubation, 1M H2SO4 was added to stop
the reaction. Promptly, the optical density value of each well was read at 450 nm using an ELISA reader (Dynex Technologies, Ashford, UK). Hepatic TNF-α and IL-1β levels were both expressed as pg mg−1 protein.
2.6. Hepatic mRNA expressions of nuclear factor kappa light chain enhancer of B cells (NF-κB), krüppel-like factor 6 (KLF-6), activator protein 1 (AP-1), interleukin-6 (IL-6), alpha-smooth muscle actin (α-SMA), collagen alpha 1 (COLα1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Total RNA was isolated from the frozen liver tissues according to manufacturer’s instructions of E.Z.N.A.TissueRNAKit (Omega Bio-Tek, Inc., Norcross, GA, USA). Semi-quantitative reverse transcription (RT-PCR) was performed with 2 µg of total RNA, 8 µL of reaction buffer, 2 µL of dNTPs, 4.8 µL of MgCl2, 4 µL of OligodT (10 µmol/L), and 200 U RTase (Promega, Madison, WI, USA) with diethyl pyrocarbonate (DEPC) H2O to reach a final volume of 40 µL at 42 °C for 1 h. After a heat inactivation, 1 µL of cDNA product was used for a PCR amplification. The appropriate primers of target genes and conditions were listed below: NFκB (GenBank no.: NM_212509.2), KLF6 (GenBank no.: NM_031642 .4), AP1 (GenBank no.: X12740.1), IL6 (GenBank no.: NM_012589.2), α-SMA (GenBank no.: NM_007392.3), COLα1 (GenBank no.: XM_003749691.1), and GAPDH (GenBank no.: FQ216837.1) as follows: NF-κB sense 5′-TGCAGCGATTCCGAAACCAG-3′, antisense 5′-AAATGGCAGTCCAGAGCAGTG-3′; KLF6 sense 5′CCTTACAGATGCTCTTGGGA-3′, antisense 5′-GGAGAAACACCTG TCACAGT-3′; AP1 sense 5′-CCGAGAGCGGTGCCTACGGCTACAG3′, antisense 5′-GACCGGCTGTGCCGCGGAGGTGAC-3′; IL6 sense 5′-TGGAGTCACAGAAGGAGTGGCTAA-3′, antisense 5′-TCTG ACCACAGTGAGGAATGTCCAC-3′; α-SMA sense 5′-GTGCTATGTAG CTCTGGACT-3′, antisense 5′-ACATCTGCTGGAAGGTAGAC-3′; COLα1 sense 5′-AACAGGACTTCTTCGGAACCAC-3′, antisense 5′CATTTGCACCACTTGTGGCTTC-3′; GAPDH sense 5′-GACCCCTT CATTGACCTCAAC-3′, antisense 5′-GGAGATGATGACCCTTTTGGC3′. The size of reaction products is as follows: for NF-κB, 287 bp; KLF6, 126 bp; AP1, 349 bp; IL6, 153 bp; α-SMA, 419 bp; COLα1, 423 bp; GAPDH, 245 bp. GAPDH was used as an internal control in all reactions. The PCR amplification was performed under conditions using a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycle, Foster City, CA, USA) under the following conditions: NF-κB and IL6: 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C; KLF6: 40 cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C; AP1: 30 cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C; α-SMA: 33 cycles at 94 °C for 1 min, 60.5 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C; COLα1 and GAPDH: 28 cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C. The final products were subjected to electrophoresis on a 2% agarose gel and detected by ethidium bromide staining then visualized by UV light.The relative expressions of the target genes were normalized using the GAPDH as an internal standard.
2.7.
Histopathological analyses
Formalin fixed liver tissues were fixed in neutral buffered formalin, and then dehydrated in graded alcohol.After impregnating
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Table 1 – Effects of D-glucuronolactone (lactone) on growth performance, and relative size of liver, heart, and kidney of the thioacetamide (TAA)-treated rats.1 Group2 Growth performance Initial body weight (g) Final body weight (g) Weight increase (g) Weight increase (%) Food intake (g/rat/day) Water intake (mL/rat/day) Relative size (g/100 g bw) Liver Heart Kidney 1 2
Control
TAA
Lactone
234.04 ± 2.80a 449.84 ± 11.11a 215.80 ± 9.90a 92.18 ± 4.11a 28.51 ± 0.71a 50.84 ± 2.86a
229.94 ± 1.57a 336.28 ± 7.80b 106.34 ± 8.22b 46.36 ± 3.68b 22.81 ± 0.63b 42.84 ± 2.44b
231.55 ± 1.30a 356.76 ± 5.39b 125.21 ± 5.45b 54.12 ± 2.42b 24.78 ± 0.69b 42.99 ± 1.29b
3.43 ± 0.06a 0.31 ± 0.01a 0.67 ± 0.01a
3.37 ± 0.07a 0.30 ± 0.01a 0.65 ± 0.01a
2.55 ± 0.07b 0.32 ± 0.01a 0.55 ± 0.03b
The data are given as mean ± SEM. Mean values in each test parameter with different superscript letters were significantly different (p < 0.05). Control: Saline (i.p) + normal distilled water (oral gavage); TAA: TAA + normal distilled water (oral gavage); Lactone: TAA+ 75 mg lactone/kg bw (oral gavage).
the tissues in xylene, they were embedded in paraffin for sectioning using a microtome. On completion of deparaffinizing with xylene, tissues were dehydrated in graded alcohol and stained with hematoxylin and eosin (H&E) as well as Masson’s trichrome staining respectively. The quantification of liver damage was assayed in area conditions of portal inflammation, intralobular degeneration, and periportal-bridging based on a histology activity index score (HAI score) (Knodell et al., 1981). The values of collagen accumulation for hepatocirrhosis were given by Metavir score, and samples were categorized into 5 levels depending on the severity from fibrosis to cirrhosis (Alric et al., 2001).
2.8.
Statistical analysis
The experiment was conducted using a completely randomized design (CRD). Data were shown as the mean ± standard error mean (SEM) and analyzed using analysis of variance (ANOVA). A significant difference between treatments at a 0.05 probability level was determined by the least significant difference (LSD) test. All statistical analyses of data were carried out using SAS software.
3.
Control group, with similar results observed in water and food intakes (Table 1). No (p > 0.05) differences between sizes of hearts in each group were recorded, but contradictorily sizes of livers and kidneys were larger (p < 0.05) in TAA-treated group (both TAA and Lactone groups) than that in Control group (Table 1). Besides, increased (p < 0.05) serum AST and ALT values were observed in TAA-treated group compared to Control group after 8 weeks of experimental period while lactone supplementation indeed reduced (p < 0.05) serum AST values but did not (p > 0.05) affect ALT values (Table 2). TAA treatment led to significantly higher (p < 0.05) liver TNF-α and IL-1β levels, but those increased values were reduced (p < 0.05) by supplementing lactone (Table 2).
Results
3.1. Effects of lactone on growth performance and serum liver damage indices Both body weights and weight increases on TAA group and Lactone group were significantly lower (p < 0.05) compared to
3.2. Effects of lactone on liver lipid peroxidation, antioxidant capacity, and enzyme activities To detect the antioxidant effect of lactone, liver GSH, TEAC and TBARS levels were measured and the results were demonstrated in Fig. 1A–C, respectively. TAA-treated rats supplemented with lactone significantly elevated (p < 0.05) GSH and TEAC levels compared to TAA group. Additionally, the TBARS levels of Lactone group were significantly lowered (p < 0.05) as expected; however, with just a slight alteration. Regarding effects on liver antioxidant enzyme activities, significantly higher (p < 0.05) SOD (Fig. 1D) and GPx (Fig. 1F) activities were observed in Lactone group compared to those in TAA group, while there were no significant changes (p > 0.05) between TAA and Lactone group on CAT activities (Fig. 1E). Notably, these results implicated that lactone supplementation shows an
Table 2 – Liver damage indices (serum AST and ALT values), as well as liver TNF-α and IL-1β levels of the thioacetamide (TAA)-treated rats.1 Group2
Control
AST (IU/L) ALT (IU/L) TNF-α (pg/mg protein) IL-1β (pg/mg protein)
133.58 ± 5.20 32.67 ± 1.83b 4.17 ± 0.35b 17.53 ± 0.89b
1 2
TAA c
Lactone
372.17 ± 7.42 121.67 ± 11.13a 5.79 ± 0.67a 26.45 ± 2.13a a
212.00 ± 6.31b 127.58 ± 5.37a 2.38 ± 0.07c 15.92 ± 0.81b
The data are given as mean ± SEM. Mean values in each test parameter with different superscript letters were significantly different (p < 0.05). Control: Saline (i.p) + normal distilled water (oral gavage); TAA: TAA + normal distilled water (oral gavage); Lactone: TAA + 75 mg lactone/kg bw (oral gavage).
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Fig. 1 – Effects of D-glucuronolactone (lactone) on liver GSH (A), TEAC (B), and TBARS (C) levels, as well as activities of SOD (C), CAT (D), and (E) GPx of the thioacetamide (TAA)-treated rats. The data are given as mean ± SEM. Different letters on data bars represent significant difference (p < 0.05). Control: Saline (i.p) + normal distilled water (oral gavage); TAA: TAA + normal distilled water (oral gavage); Lactone: TAA + 75 mg lactone/kg bw (oral gavage).
antioxidant effect on an oxidative stress of livers induced by TAA treatment.
3.3. Effects of lactone on liver fibrosis and inflammatory responses To evaluate the molecular mechanism of anti-fibrosis and antiinflammatory effects of lactone, expressions of related genes were detected. Regarding gene expressions of inflammation and fibrosis related factors, higher (p < 0.05) gene expressions of NFκB, KLF6, AP1, IL6, α-SMA, and COLα1 in livers were observed in TAA group than those in Control group (Fig. 2). However, the lactone supplementation downregulated (p < 0.05) those gene expressions, except COLα where only a lower tendency was observed. Via H&E staining, results of histopathological examination in livers indicated inflammatory cell infiltration around the central vein (CV) in TAA group (Fig. 3A). Paradoxically, lactone supplementation alleviated infiltration of lymphocytes to reach an ameliorative effect, corresponding to the consequences of HAI scores (more detailed) (Fig. 3B). Besides, an obvious collagen accumulation (arrows) in liver sections of TAA group was observed via Masson’s trichrome staining (Fig. 3A) which was related to higher scores of METAVIR fibrosis (Fig. 3C). Conversely, lactone supplementation markedly reduced contents of collagen in livers.
4.
Discussion
TAA is a hepatotoxin which is widely used for inducing liver fibrosis. Metabolism of TAA which generates reactive
compounds increasing oxidative stress underlies the damage of livers, in parallel with releasing AST and ALT from liver (Túnez et al., 2005), corresponding to our data. The decreased body weight and larger size of livers of experimental rats treated with TAA might be relevant to liver damage, doing harm to body as well as ECM accumulation (Chen et al., 2011). TAA-treated rats showed signs of kidney injury coinciding with cell distention (Barker & Smuckler, 1974). It was reported that administration of TAA caused larger kidneys (Korsrud, Grice, Goodman, Knipfel, & McLaughlan, 1973), similar to our findings. Those consequences affected by TAA were ameliorated by lactone treatment, except ALT, indicating that lactone has potential effects on hepatoprotection. The metabolites of TAA like TASO 2 can covalently bind to proteins forming acetylimidolysine derivatives. This leads to increase in oxidative stress which evokes the enzymatic and non-enzymatic antioxidant system against accumulation of ROS and lipid peroxidation, further preventing liver from severe dysfunction (MatÉs, Pérez-Gómez, & De Castro, 1999). Enzymatic system encompasses several enzymes like CAT and GPx (Jia et al., 2011), which catalyzes hydrogen peroxide transforming to H2O and O2. SOD is also one of the antioxidant enzymes responsible for transferring superoxide radicals to less activated compounds. We found that liver CAT and SOD activities were facilitated to maintain the balance of ROS production in TAA-treated rats. Thus, the produced ROS may have turned into nontoxic compounds. However, activities of GPx and GSH contents in livers were decreased, accompanied with higher TBARS values after a TAA treatment, implicating that overproduction of ROS could hardly be removed by those mentioned under TAA
journal of functional foods 14 (2015) 154–162
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Fig. 2 – Effects of D-glucuronolactone (lactone) on gene expressions of KLF6, NFκB, COLα1, AP1, α-SMA, and IL6 in the livers of thioacetamide (TAA)-treated rats. The data are given as mean ± SEM. Different letters on data bars of each target gene represent significant difference (p < 0.05). The value of hepatic KLF6, NFκB, COLα1, AP1, α-SMA, and IL6 mRNA expressions were normalized to the value of GAPDH and values of rats in TAA and Lactone groups were expressed relatively to the average values for rats in the Control group, which was set to 1.0. Control: Saline (i.p) + normal distilled water (oral gavage); TAA: TAA + normal distilled water (oral gavage); Lactone: TAA+ 75 mg lactone/kg bw (oral gavage).
treatment. Similar results have been reported in other oxidative stress-induced models, and supplementation of natural ingredients, i.e. Antrodia camphorata (Chou, Chang, Hung, Chen, & Chiu, 2013) and litchi (Litchi chinensis Sonn.) flower–water extract (Chang et al., 2013) which present hepatoprotective effects via enhancing antioxidant capacities. Hence, lactone attenuated the increased oxidative stress; that is, increasing SOD and GPx activities as well as GSH contents and TEAC levels, thus decreasing TBARS values. Little evidence indicated that lactone has antioxidant capacity. Nevertheless, metabolites of lactone are able to help to excrete toxin by conjugating with xenobiotics to form more hydrophilic compounds through Phase II detoxification and further reduce the oxidative stress (Gao, Dinkova-Kostova, & Talalay, 2001). This indirectly antioxidant effect may underlie the relatively higher activities of SOD and GPx, and further provide a hepatoprotective effect. Liver fibrosis, the advanced symptom of excessive ROS targeting on liver, features accumulation of collagen secreted by HSCs, which is activated by inflammatory cells (Bataller & Brenner, 2005). The inflammatory response causes stimulation of related genes (IL6, IL-1β and TNF-α) and further neutrophil infiltration, which can be observed in our result (Fig. 3A). The transcriptional factors like KLF6, NFκB, and AP-1 are also involved in both inflammation and HSC activation. The inhibition of NFκB, an essential role in proinflammatory response activated by TNFα, has received much attention in antioxidant and anti-inflammatory effects. An induction of NFκB associated with activated HSC often relates to liver damage because it imposes a constraint on HSC apoptosis, further leading to aggregating hepatic fibrosis (Mann & Smart, 2002). In other words, those transcriptional factors and cytokines have a relatively positive correlation with liver fibrosis, and in accordance with our results indicating that liver damage had been
induced by a TAA treatment through an elevation of those factors. The activated HSCs transfer into myofibroblast-like cells, with expressions of COLα1 as well as α-SMA and trigger a generation of ECM, consisting mainly of collagen types I and III after hepatic necrosis in order to conduct wound-healing response on injured liver to attain liver regeneration (Stalnikowitz & Weissbrod, 2003). Thus, the dissemination of ECM in liver depends on where damage occurred (Bataller & Brenner, 2005) and often aggregated in portal tracts, around hepatocytes and bile ducts (Pinzani & Rombouts, 2004). Those features of liver fibrosis have prospect to be reversible (Bataller & Brenner, 2005) if continued damage is prohibited by functional ingredients. Based on histopathological findings from this study, lactone exhibited a capacity of anti-fibrosis which apparently decreased collagen accumulation (Fig. 3A) and it is probably caused by a down-regulation of COLα1. Lower expression of α-SMA in Lactone group also indicated the less severe fibrosis. The aforementioned capacity of lactone, including anti-inflammation and anti-fibrosis effects, induces a benign cycle to promote each other leading to the amelioration of liver injury. Lack of evidence in lactone supports the assumption that lactone can ameliorate liver damage; however, according to our results, liver fibrosis of rats induced by TAA can be attenuated by supplementing lactone. Hence, we assumed that those effects are attributed to lactone itself and its metabolites. In chronic liver fibrosis model built by carbon tetrachloride (CCl4) injection on mice, lactone attenuated the sharp rise of AST and ALT which demonstrated the potential hepatoprotective capacity of lactone (Chang & Chien, 2011). Furthermore, some energy drinks that contain lactone claim that together with other related derivatives especially glucaric acid they have anticancer effects via inhibition of β-glucuronidase (Higgins, Tuttle, & Higgins, 2010). The antioxidant effects may be derived from
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Fig. 3 – Effects of D-glucuronolactone (lactone) on (A) gross appearance, H&E staining, and Masson trichrome staining, (B) histology activity index score (HAI score), and (C) Metavir score of the liver section in the thioacetamide (TAA)-treated rats. CV is an abbreviation of central vein.
ascorbic acid synthesized from one of the metabolites of lactone-L-gulunolactone through gulunolactone oxidase which is deficient in humans (Marsh, 1963). Bastway Ahmed, Hasona, and Selemain (2010) also suggested that ascorbic acid had the ability to attenuate TAA-induced liver injury relying on inhibition of elevated TBARS values and relative increase of GSH contents. Another metabolite of lactone – glucuronic acid participates in Phase II detoxification of livers via a conjugation with xenobiotics to prevent liver from further damage caused by drug or other toxins. The elevated Phase II activity by glucuronic acid may recover the imbalance of Phase I and Phase II which closely relates to cancer induction (Wilkinson & Clapper, 1997). It has also been reported that the kombucha
tea containing glucuronic acid can provide hepatoprotective effects on carbon tetrachloride-induced liver injury (Murugesan et al., 2009). Those investigations support our findings of lactone as a potential functional ingredient for alleviating the development of liver fibrosis, and it may be correlated with itself or its metabolites.
5.
Conclusion
In conclusion (Fig. 4), lactone can attenuate TAA-induced liver fibrosis via two major pathways including anti-fibrosis
journal of functional foods 14 (2015) 154–162
161
Fig. 4 – Schematic representation of protective mechanism by which D-glucuronolactone (lactone) alleviates thioacetamide (TAA)-induced liver damage.
(reductions of collagen accumulation in liver and related gene expressions), and anti-inflammation (attenuations of elevated ALT, cytokines and expressions of related genes). Lactone provides great antioxidant capacity (potentiation of SOD and GPx activities) as well and further helps to prevent an advanced liver injury. This investigation implicated a novel view on function of lactone, and it could be a fundamental functional ingredient for prevention of TAA-induced liver fibrosis.
Acknowledgements We acknowledge the funding of this research by the National Science Council Taiwan (R.O.C.) (Project: NSC 101-2815-C-002152-B) and a kind offer of D-glucuronolactone (C6H8O6, lactone) from Forever Chemical Co., Ltd. (Tauyan County, Taiwan).
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Ministry of Health and Welfare, Executive Yuan, Taiwan. (2014). 2013 statistics of causes of death (created date: 2014/ 07/31).http://www.mohw.gov.tw/EN/Ministry/Statistic. aspx?f_list_no=474&fod_list_no=5044. Difeo, A., Martignetti, J. A., & Narla, G. (2009). The role of KLF6 and its splice variants in cancer therapy. Drug Resistance Updates, 12, 1–7. Finnegan, D. (2003). The health effects of stimulant drinks. Nutrition Bulletin, 28, 147–155. Gao, X., Dinkova-Kostova, A. T., & Talalay, P. (2001). Powerful and prolonged protection of human retinal pigment epithelial cells, keratinocytes, and mouse leukemia cells against oxidative damage: The indirect antioxidant effects of sulforaphane. Proceedings of the National Academy of Sciences of the United States of America, 98, 15221–15226. Hanausek, M., Walaszek, Z., & Slaga, T. J. (2003). Detoxifying cancer causing agents to prevent cancer. Integrative Cancer Therapies, 2, 139–144. Higgins, J. P., Tuttle, T. D., & Higgins, C. L. (2010). Energy beverages: Content and safety. Mayo Clinic Proceedings, 85, 1033–1041. Jia, X. Y., Zhang, Q. A., Zhang, Z. Q., Wang, Y., Yuan, J. F., Wang, H. Y., & Zhao, D. (2011). Hepatoprotective effects of almond oil against carbon tetrachloride induced liver injury in rats. Food Chemistry, 125, 673–678. Kim, H. Y., Kim, S. J., Kim, K. N., Lee, S. G., & Lee, S. M. (2011). Protective effect of HV-P411, an herbal mixture, on carbon tetrachloride-induced liver fibrosis. Food Chemistry, 124, 248– 253. Knodell, R. G., Kamal, G. I., William, C. B., Thomas, S. C., Robert, C. N. K., Thomas, W. K., & Jerome, W. (1981). Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology (Baltimore, Md.), 1, 431–435. Korsrud, G. O., Grice, H. G., Goodman, T. K., Knipfel, J. E., & McLaughlan, J. M. (1973). Sensitivity of several serum enzymes for the detection of thioacetamide-, dimethylnitrosamine- and diethanolamine-induced liver damage in rats. Toxicology and Applied Pharmacology, 26, 299– 313.
Mann, D. A., & Smart, D. E. (2002). Transcriptional regulation of hepatic stellate cell activation. Gut, 50, 891–896. Marsh, C. A. (1963). Metabolism of D-glucuronolactone in mammalian systems 2. Conversion of D-glucuronolactone into D-glucaric acid by tissue preparations. Biochemical Journal, 87, 82–90. MatÉs, J. M., Pérez-Gómez, C., & De Castro, I. N. (1999). Antioxidant enzymes and human diseases. Clinical Biochemistry, 32, 595–603. Mehendale, H. M. (2005). Tissue repair: An important determinant of final outcome of toxicant-induced injury. Toxicologic Pathology, 33, 41–51. Murugesan, G. S., Sathishkumar, M., Jayabalan, R., Binupriya, A. R., Swaminathan, K., & Yun, S. E. (2009). Hepatoprotective and curative properties of Kombucha tea against carbon tetrachloride-induced toxicity. Journal of Microbiology and Biotechnology, 19, 397–402. Pinzani, M., & Rombouts, K. (2004). Liver fibrosis: From the bench to clinical targets. Digestive and Liver Disease, 36, 231– 242. Scientific Opinion of the Panel on Food Additives and Nutrient Sources Added to Food (2009). The use of taurine and D-glucurono-γ-lactone as constituents of the so called “energy” drinks. The EFSA Journal, 935, 1–31. Stalnikowitz, D. K., & Weissbrod, A. B. (2003). Liver fibrosis and inflammation. A review. Annals of Hepatology, 2, 159–163. Túnez, I., Muñoz, M. C., Villavicencio, M. A., Medina, F. J., de Prado, E. P., Espejo, I., Barcos, M., Salcedo, M., Feijóo, M., & Montilla, P. (2005). Hepato-and neurotoxicity induced by thioacetamide: Protective effects of melatonin and dimethylsulfoxide. Pharmacological Research, 52, 223–228. Walaszek, Z., Szemraj, J., Narog, M., Adams, A. K., Kilgore, J., Sherman, U., & Hanause, M. (1997). Metabolism, uptake, and excretion of a D-glucaric acid salt and its potential use in cancer prevention. Cancer Detection and Prevention, 21, 178 – 190. Wilkinson, J., & Clapper, M. L. (1997). Detoxication enzymes and chemoprevention. Experimental Biology and Medicine, 216, 192 – 200.
Available online at www.sciencedirect.com
ScienceDirect j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j ff
Ameliorative effects of D-glucuronolactone on oxidative stress and inflammatory/fibrogenic responses in livers of thioacetamide-treated rats Po-Ju Chen a, Chih-Hsien Chiu a, Jung-Kai Tseng b, Kou-Tai Yang a, Yi-Chen Chen a,* a b
Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan School of Optometry, Chung Shan Medical University, Taichung 402, Taiwan
A R T I C L E
I N F O
A B S T R A C T
Article history:
D-Glucuronolactone (C6H8O6, lactone), naturally found in plant gums, is commercially ac-
Received 7 August 2014
claimed for its hepatoprotective effects. This study was to investigate whether lactone can
Received in revised form 14 January
attenuate thioacetamide (TAA)-induced liver fibrosis in a rat model. Results showed that
2015
lactone supplementation (75 mg kg−1 bw glucuronolactone) alleviated AST values in TAA-
Accepted 21 January 2015
intraperitoneally-injected rats (100 mg kg−1 bw TAA) and increased antioxidant capacity of
Available online
liver via elevations of antioxidant enzymes activities [superoxide dismutase (SOD) and glutathione peroxidase (GPx)], glutathione (GSH) and trolox equivalent antioxidative capacity
Keywords:
(TEAC) levels (p < 0.05). Down-regulated (p < 0.05) expression of inflammation including
Anti-inflammation
interleukin-6 (IL-6), nuclear factor-kappa B (NF-κB), activator protein 1 (AP-1), krüppel-like
Antioxidant
factor 6 (KLF-6), and fibrosis related fibrotic factors, i.e., alpha smooth muscle actin (α-SMA),
D-glucuronolactone
and collagen alpha1 (I) (COLα1) through lactone supplementation underlay the lower col-
Liver fibrosis
lagen contents and less severe liver damage on histopathology observations. Therefore,
Thioacetamide
hepatoprotection of lactone against TAA-induced liver fibrosis can be attributed to the amelioration of oxidative stress and inflammation. © 2015 Elsevier Ltd. All rights reserved.
1.
Introduction
In recent years, there has been a gradual rise in the number of people suffering from liver diseases, mainly caused by alcohol, toxins, drugs, viruses, and abnormal dietary habits. In Taiwan, chronic liver disease and cirrhosis ranked sixth among leading causes of death in 2013 (Ministry of Health and Welfare, Executive Yuan, Taiwan, 2014). Liver fibrosis is often related to escalating reactive oxygen species (ROS) in the body. Excessive ROS results in an inflammation of hepatocytes and cytokine
secretion, and activates hepatic stellate cells (HSCs) into myofibroblast-like cells with secreting extracellular matrix (ECM) proteins at the site of tissue repairs, further leading to hepatic fibrosis (Kim, Kim, Kim, Lee, & Lee, 2011). The characteristic of hepatic fibrosis features a progressive accumulation of ECM proteins, produced by activated HSCs. It has been indicated that HSCs are associated with inflammation-related genes (e.g., IL1β, IL6, and TNF-α) (Bataller & Brenner, 2005) and activated by many transcriptional factors like nuclear factor-kappa B (NFκB), activator protein 1 (AP-1), and krüppel-like factor 6 (KLF6), as well as alpha smooth muscle actin (α-SMA) and collagen
* Corresponding author. Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan. Tel.: +886 2 33664180; fax: +886 2 27324070. E-mail address: [email protected] (Y.-C. Chen). http://dx.doi.org/10.1016/j.jff.2015.01.026 1756-4646/© 2015 Elsevier Ltd. All rights reserved.
journal of functional foods 14 (2015) 154–162
alpha 1 (COLα1) (Chen et al., 2011; Difeo, Martignetti, & Narla, 2009; Mann & Smart, 2002). Based on potential reversibility of hepatic fibrosis, researchers have paid much attention to antifibrotic studies. Thioacetamide (CH3C(S)NH2, TAA) was found to cause liver damage, and hence, used in a number of research on liverrelated studies as a hepatotoxicant to induce acute and chronic liver diseases due to several advantages as follows: 1) high specificity for liver and regiospecificity for perivenous area; 2) relatively short half-life for a rapid induction of acute liver damage; 3) large window of time between its necrogenic effects and liver failure for better observation (Mehendale, 2005). TAA was found to be toxic after being metabolized (Chieli & Malvaldi, 1984). TAA is metabolically converted to thioacetamide-Soxide (TASO), acetamide and sulfate through microsomal mixedfunction oxidase system. TASO leads to centrilobular hepatic necrosis. A further metabolism causes biotransformation of TASO to thioacetamide-S, S-dioxide (TASO2), covalently binding to proteins with forming acetylimidolysine derivatives which act as hepatotoxic compounds. Originally, D-glucuronolactone (C6H8O6, lactone), also called D-glucuronic acid gamma-lactone, is a natural metabolite of glucose in animal livers and also found in plant gums with a complex form (Finnegan, 2003). It is commercially produced by nitric acid oxidation of starch and has been widely used as a common ingredient in energy drinks and also commercially acclaimed for its function of providing energy (Scientific Opinion of the Panel on Food Additives and Nutrient Sources Added to Food, 2009). Despite using lactone as a food ingredient, the safety of lactone has been debated. Ahrens, Douglass, Flynn, and Ward (1987) also indicated that there are no significant influences on growth performance of rats receiving the lactone. Scientific Opinion of the Panel on Food Additives and Nutrient Sources Added to Food (2009) in European Food Safety Authority reported that Scientific Committees on Food has examined a long-term toxicity of the lactone on both rat and hamster models. There are some conclusions below: 1) No significant treatment-related side effects on animals except kidney at high doses (600 and 1000 mg kg−1 bw day−1); 2) No-observedadverse-effect level (NOAEL) of the lactone in this experiment was 1000 mg kg−1 bw day−1. Hence, lactone supplementation seems to be safe at least with controllable dose. Lactone, in mammals, can be hydrolyzed to D-glucuronic acid, and converted into L-gulonolactone reversibly, as well as D-glucaric acid (Marsh, 1963). It has been indicated that D-glucuronic acid plays a crucial role in Phase II reaction of drug metabolism by conjugating with xenobiotics and other foreign compounds to form more hydrophilic substances, which are easily excreted in the urine (Hanausek, Walaszek, & Slaga, 2003). In addition, D-glucaric acid is normally observed in mammalian urine without toxicity, and its derivatives are also demonstrated to prevent breast, prostate, and colon cancer (Walaszek et al., 1997). However, few studies on ameliorative effects or the molecular mechanism of lactone on liver fibrosis have been available until now. Therefore, the current study was to induce a liver fibrosis of rats by an injection of TAA. Then, the ameliorative effects of lactone on TAA-induced liver fibrosis were investigated as the following aspects: 1) antioxidative capacities, 2) anti-inflammation, and 3) anti-fibrosis.
2.
Materials and methods
2.1.
Animals and treatments
155
The animal use and protocol were reviewed and approved by National Taiwan University Animal Care Committee (IACUU No.: 100-15). Thirty-six male Wistar rats of 5 weeks in age were purchased from BioLASCO Taiwan Co., Ltd (Taipei, Taiwan), and acclimatized for one week before commencement of the experiment. Two rats were housed in each cage in an animal room at 22 ± 2 °C with a 12/12 h light–dark cycle, and randomly assigned to one of the following groups (n = 12 per group): 1) Control group: sterilized saline (intraperitoneally injection, i.p.) + 0.5 mL normal distilled water (oral gavage); 2) TAA group: 100 mg kg−1 bw TAA (i.p.) + 0.5 mL normal distilled water (oral gavage); 3) Lactone group: 100 mg kg−1 bw TAA (i.p.) + 75 mg lactone kg−1 bw in 0.5 mL normal distilled water (oral gavage). Animal experimental period lasted for 8 weeks with injections of TAA three times weekly (Monday, Wednesday, and Friday) and oral gavage daily. Chow diets containing 487 g carbohydrate, 239 g protein, 50 g fat, 51 g fiber, and 70 g ash kg−1 (Laboratory Rodent Diet 5001, PMI Nutrition International/ Purina Mills LLC, Richmond, IN, USA) with water were provided ad libitum. Lactone was kindly offered from Forever Chemical Co., Ltd. (Tauyan County, Taiwan), and TAA was purchased from Sigma Co. (St. Louis, MO, USA). Average daily food and water intakes per cage were calculated as volumes of food and water intakes on per rat daily basis, respectively. All rats were fasted overnight before euthanized by CO2 in the final day of experiment. Liver, heart, and kidney from each rat were removed, weighted and then stored at −80 °C for further analyses.
2.2.
Determination of serum liver damage indices
Blood samples were collected via abdominal aorta, and centrifuged at 3000 × g for 10 min to obtain sera, stored at −80 °C. Liver damage indices [alanine aminotransferase (ALT) and aspartate aminotransferase (AST)] were analyzed using commercial enzymatic kits with an ESPOTCHEMTMEZ SP4430 biochemistry analyzer (ARKRAY Inc., Kyoto, Japan).
2.3.
Preparation of liver homogenate
A 0.5 g sample of liver was homogenized on ice with 4.5 mL phosphate buffer saline (PBS, pH 7.0, containing 0.25 M sucrose). After a centrifugation (12,000 × g for 30 min) at 4 °C, supernatant was collected for further analyses. Protein levels in supernatant were measured by a Bio-Rad protein assay kit (Cat. #: 500-0006, Bio-Rad Laboratories, Inc., Hercules, CA, USA) using bovine serum albumin as a standard.
2.4.
Determination of antioxidant capacity in livers
The liver thiobarbituric-acid-reactive-substances (TBARS), glutathione (GSH), trolox equivalent antioxidative capacity (TEAC) levels, as well as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were determined to represent hepatic antioxidant capacities. Lipid peroxidation (TBARS) was determined by adding TBA solution
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and trichloroacetic acid–HCl (TCA–HCl) solution to homogenate. Mixture was reacted at 95 °C for 30 min, followed by centrifugation at 4000 × g for 15 min and then read at 535 nm. For measuring GSH contents, homogenates were reacted with TCA and centrifuged at 9000 × g for 5 min to obtain supernatant. Supernatant reacted with 2,2-dithio-bis-nitrobenzoic acid (DTNB) for 5 min, and then read at 412 nm.The TBARS and GSH values were calculated by taking the extinction coefficients of MDA to be 1.56 × 105 M−1 cm−1 at 532 nm and 2-nitro-5-thiobenzoic acid to be 1.36 × 104 M−1 cm−1 at 412 nm, respectively. In the assay of TEAC, homogenates were mixed with ABTS, peroxidase, as well as H2O2 under a dark room, and then measured at 734 nm after 10 min. The decrease in absorption at 734 nm was used to calculate the TEAC value. A standard curve was plotted for trolox on scavenging ABTS+ capacity and was used to calculated as the TEAC. For the purpose of determining SOD, homogenate added with Tris–HCl buffer (50 mM, pH 8.2) was prepared in advance and then the mixture was reacted with pyrogallol (0.2 mM) followed by measurement of the absorbance at 420 nm. One unit of SOD activity was defined as the amount of enzyme that inhibited the autoxidation of pyrogallol by 50%. CAT activity was measured by mixing homogenate with H2O2, and calculated by taking the extinction coefficient of H2O2 to be 39.5 M−1 cm−1 at 240 nm. One unit of CAT was expressed as the amount of enzyme that decomposes 1 mole H2O2 per minute at 25 °C. The measurement of GPx activity was briefly described as following. Homogenate was mixed with a reaction solution containing 100 mM PBS (pH 7.0), 10 mM EDTA, 10 mM NaN3, 2 mM NAPDH, 10 mM GSH, and 1 U/mL GSH reductase. After 5 min, H2O2 (2.5 mM) was then added and measured at 340 nm in a 3-min interval. Hepatic GPx activity was calculated by taking the extinction coefficient of NADPH to be 6.22 × 106 nM−1 cm−1. The liver TBARS, GSH, and TEAC levels were demonstrated as nmole MDA equivalent mg−1 protein, nmole mg−1 protein, and nmole mg−1 protein, respectively. The liver SOD, CAT, and GPx were demonstrated as unit mg−1 protein, unit mg−1 protein, and nmole NADPH oxidized min−1 mg−1 protein, respectively.
2.5.
Determination the level of TGF-β and IL-1β in livers
The hepatic TNF-α and IL-1β levels were measured by using enzyme-linked immunosorbent assay (ELISA) and were conducted according to the commercial manufacturer’s instructions (TNF-α and IL-1β kits, eBioscience Inc., San Diego, CA, USA). An aliquot of liver containing 100–200 ng of protein was extracted in the liver homogenate. First, the capture antibody (antiTNF-α and anti-IL-1β) was diluted with a coating buffer as a working solution. Each well of flat-bottom 96-well ELISA plates was coated with working solution and then incubated at 4 °C overnight. Thereafter, the plates were rinsed three times with washing buffer (1X PBS with 0.05% Tween-20). Then, the reagent diluent were added into plates and reacted for 1 h and rinsed as foregoing way. After prepared samples were loaded and incubated for 2 h, the plates were rinsed in the same way as described previously, followed by adding assay diluent reagent, incubation for 2 h and rinsed again. The working dilution of streptavidin–HRP was added to each well and then incubated for 20 min. Finally, the mixture was added with substrate solution. After a 30-min incubation, 1M H2SO4 was added to stop
the reaction. Promptly, the optical density value of each well was read at 450 nm using an ELISA reader (Dynex Technologies, Ashford, UK). Hepatic TNF-α and IL-1β levels were both expressed as pg mg−1 protein.
2.6. Hepatic mRNA expressions of nuclear factor kappa light chain enhancer of B cells (NF-κB), krüppel-like factor 6 (KLF-6), activator protein 1 (AP-1), interleukin-6 (IL-6), alpha-smooth muscle actin (α-SMA), collagen alpha 1 (COLα1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Total RNA was isolated from the frozen liver tissues according to manufacturer’s instructions of E.Z.N.A.TissueRNAKit (Omega Bio-Tek, Inc., Norcross, GA, USA). Semi-quantitative reverse transcription (RT-PCR) was performed with 2 µg of total RNA, 8 µL of reaction buffer, 2 µL of dNTPs, 4.8 µL of MgCl2, 4 µL of OligodT (10 µmol/L), and 200 U RTase (Promega, Madison, WI, USA) with diethyl pyrocarbonate (DEPC) H2O to reach a final volume of 40 µL at 42 °C for 1 h. After a heat inactivation, 1 µL of cDNA product was used for a PCR amplification. The appropriate primers of target genes and conditions were listed below: NFκB (GenBank no.: NM_212509.2), KLF6 (GenBank no.: NM_031642 .4), AP1 (GenBank no.: X12740.1), IL6 (GenBank no.: NM_012589.2), α-SMA (GenBank no.: NM_007392.3), COLα1 (GenBank no.: XM_003749691.1), and GAPDH (GenBank no.: FQ216837.1) as follows: NF-κB sense 5′-TGCAGCGATTCCGAAACCAG-3′, antisense 5′-AAATGGCAGTCCAGAGCAGTG-3′; KLF6 sense 5′CCTTACAGATGCTCTTGGGA-3′, antisense 5′-GGAGAAACACCTG TCACAGT-3′; AP1 sense 5′-CCGAGAGCGGTGCCTACGGCTACAG3′, antisense 5′-GACCGGCTGTGCCGCGGAGGTGAC-3′; IL6 sense 5′-TGGAGTCACAGAAGGAGTGGCTAA-3′, antisense 5′-TCTG ACCACAGTGAGGAATGTCCAC-3′; α-SMA sense 5′-GTGCTATGTAG CTCTGGACT-3′, antisense 5′-ACATCTGCTGGAAGGTAGAC-3′; COLα1 sense 5′-AACAGGACTTCTTCGGAACCAC-3′, antisense 5′CATTTGCACCACTTGTGGCTTC-3′; GAPDH sense 5′-GACCCCTT CATTGACCTCAAC-3′, antisense 5′-GGAGATGATGACCCTTTTGGC3′. The size of reaction products is as follows: for NF-κB, 287 bp; KLF6, 126 bp; AP1, 349 bp; IL6, 153 bp; α-SMA, 419 bp; COLα1, 423 bp; GAPDH, 245 bp. GAPDH was used as an internal control in all reactions. The PCR amplification was performed under conditions using a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycle, Foster City, CA, USA) under the following conditions: NF-κB and IL6: 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C; KLF6: 40 cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C; AP1: 30 cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C; α-SMA: 33 cycles at 94 °C for 1 min, 60.5 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C; COLα1 and GAPDH: 28 cycles at 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 2 min followed by 10 min at 72 °C. The final products were subjected to electrophoresis on a 2% agarose gel and detected by ethidium bromide staining then visualized by UV light.The relative expressions of the target genes were normalized using the GAPDH as an internal standard.
2.7.
Histopathological analyses
Formalin fixed liver tissues were fixed in neutral buffered formalin, and then dehydrated in graded alcohol.After impregnating
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Table 1 – Effects of D-glucuronolactone (lactone) on growth performance, and relative size of liver, heart, and kidney of the thioacetamide (TAA)-treated rats.1 Group2 Growth performance Initial body weight (g) Final body weight (g) Weight increase (g) Weight increase (%) Food intake (g/rat/day) Water intake (mL/rat/day) Relative size (g/100 g bw) Liver Heart Kidney 1 2
Control
TAA
Lactone
234.04 ± 2.80a 449.84 ± 11.11a 215.80 ± 9.90a 92.18 ± 4.11a 28.51 ± 0.71a 50.84 ± 2.86a
229.94 ± 1.57a 336.28 ± 7.80b 106.34 ± 8.22b 46.36 ± 3.68b 22.81 ± 0.63b 42.84 ± 2.44b
231.55 ± 1.30a 356.76 ± 5.39b 125.21 ± 5.45b 54.12 ± 2.42b 24.78 ± 0.69b 42.99 ± 1.29b
3.43 ± 0.06a 0.31 ± 0.01a 0.67 ± 0.01a
3.37 ± 0.07a 0.30 ± 0.01a 0.65 ± 0.01a
2.55 ± 0.07b 0.32 ± 0.01a 0.55 ± 0.03b
The data are given as mean ± SEM. Mean values in each test parameter with different superscript letters were significantly different (p < 0.05). Control: Saline (i.p) + normal distilled water (oral gavage); TAA: TAA + normal distilled water (oral gavage); Lactone: TAA+ 75 mg lactone/kg bw (oral gavage).
the tissues in xylene, they were embedded in paraffin for sectioning using a microtome. On completion of deparaffinizing with xylene, tissues were dehydrated in graded alcohol and stained with hematoxylin and eosin (H&E) as well as Masson’s trichrome staining respectively. The quantification of liver damage was assayed in area conditions of portal inflammation, intralobular degeneration, and periportal-bridging based on a histology activity index score (HAI score) (Knodell et al., 1981). The values of collagen accumulation for hepatocirrhosis were given by Metavir score, and samples were categorized into 5 levels depending on the severity from fibrosis to cirrhosis (Alric et al., 2001).
2.8.
Statistical analysis
The experiment was conducted using a completely randomized design (CRD). Data were shown as the mean ± standard error mean (SEM) and analyzed using analysis of variance (ANOVA). A significant difference between treatments at a 0.05 probability level was determined by the least significant difference (LSD) test. All statistical analyses of data were carried out using SAS software.
3.
Control group, with similar results observed in water and food intakes (Table 1). No (p > 0.05) differences between sizes of hearts in each group were recorded, but contradictorily sizes of livers and kidneys were larger (p < 0.05) in TAA-treated group (both TAA and Lactone groups) than that in Control group (Table 1). Besides, increased (p < 0.05) serum AST and ALT values were observed in TAA-treated group compared to Control group after 8 weeks of experimental period while lactone supplementation indeed reduced (p < 0.05) serum AST values but did not (p > 0.05) affect ALT values (Table 2). TAA treatment led to significantly higher (p < 0.05) liver TNF-α and IL-1β levels, but those increased values were reduced (p < 0.05) by supplementing lactone (Table 2).
Results
3.1. Effects of lactone on growth performance and serum liver damage indices Both body weights and weight increases on TAA group and Lactone group were significantly lower (p < 0.05) compared to
3.2. Effects of lactone on liver lipid peroxidation, antioxidant capacity, and enzyme activities To detect the antioxidant effect of lactone, liver GSH, TEAC and TBARS levels were measured and the results were demonstrated in Fig. 1A–C, respectively. TAA-treated rats supplemented with lactone significantly elevated (p < 0.05) GSH and TEAC levels compared to TAA group. Additionally, the TBARS levels of Lactone group were significantly lowered (p < 0.05) as expected; however, with just a slight alteration. Regarding effects on liver antioxidant enzyme activities, significantly higher (p < 0.05) SOD (Fig. 1D) and GPx (Fig. 1F) activities were observed in Lactone group compared to those in TAA group, while there were no significant changes (p > 0.05) between TAA and Lactone group on CAT activities (Fig. 1E). Notably, these results implicated that lactone supplementation shows an
Table 2 – Liver damage indices (serum AST and ALT values), as well as liver TNF-α and IL-1β levels of the thioacetamide (TAA)-treated rats.1 Group2
Control
AST (IU/L) ALT (IU/L) TNF-α (pg/mg protein) IL-1β (pg/mg protein)
133.58 ± 5.20 32.67 ± 1.83b 4.17 ± 0.35b 17.53 ± 0.89b
1 2
TAA c
Lactone
372.17 ± 7.42 121.67 ± 11.13a 5.79 ± 0.67a 26.45 ± 2.13a a
212.00 ± 6.31b 127.58 ± 5.37a 2.38 ± 0.07c 15.92 ± 0.81b
The data are given as mean ± SEM. Mean values in each test parameter with different superscript letters were significantly different (p < 0.05). Control: Saline (i.p) + normal distilled water (oral gavage); TAA: TAA + normal distilled water (oral gavage); Lactone: TAA + 75 mg lactone/kg bw (oral gavage).
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Fig. 1 – Effects of D-glucuronolactone (lactone) on liver GSH (A), TEAC (B), and TBARS (C) levels, as well as activities of SOD (C), CAT (D), and (E) GPx of the thioacetamide (TAA)-treated rats. The data are given as mean ± SEM. Different letters on data bars represent significant difference (p < 0.05). Control: Saline (i.p) + normal distilled water (oral gavage); TAA: TAA + normal distilled water (oral gavage); Lactone: TAA + 75 mg lactone/kg bw (oral gavage).
antioxidant effect on an oxidative stress of livers induced by TAA treatment.
3.3. Effects of lactone on liver fibrosis and inflammatory responses To evaluate the molecular mechanism of anti-fibrosis and antiinflammatory effects of lactone, expressions of related genes were detected. Regarding gene expressions of inflammation and fibrosis related factors, higher (p < 0.05) gene expressions of NFκB, KLF6, AP1, IL6, α-SMA, and COLα1 in livers were observed in TAA group than those in Control group (Fig. 2). However, the lactone supplementation downregulated (p < 0.05) those gene expressions, except COLα where only a lower tendency was observed. Via H&E staining, results of histopathological examination in livers indicated inflammatory cell infiltration around the central vein (CV) in TAA group (Fig. 3A). Paradoxically, lactone supplementation alleviated infiltration of lymphocytes to reach an ameliorative effect, corresponding to the consequences of HAI scores (more detailed) (Fig. 3B). Besides, an obvious collagen accumulation (arrows) in liver sections of TAA group was observed via Masson’s trichrome staining (Fig. 3A) which was related to higher scores of METAVIR fibrosis (Fig. 3C). Conversely, lactone supplementation markedly reduced contents of collagen in livers.
4.
Discussion
TAA is a hepatotoxin which is widely used for inducing liver fibrosis. Metabolism of TAA which generates reactive
compounds increasing oxidative stress underlies the damage of livers, in parallel with releasing AST and ALT from liver (Túnez et al., 2005), corresponding to our data. The decreased body weight and larger size of livers of experimental rats treated with TAA might be relevant to liver damage, doing harm to body as well as ECM accumulation (Chen et al., 2011). TAA-treated rats showed signs of kidney injury coinciding with cell distention (Barker & Smuckler, 1974). It was reported that administration of TAA caused larger kidneys (Korsrud, Grice, Goodman, Knipfel, & McLaughlan, 1973), similar to our findings. Those consequences affected by TAA were ameliorated by lactone treatment, except ALT, indicating that lactone has potential effects on hepatoprotection. The metabolites of TAA like TASO 2 can covalently bind to proteins forming acetylimidolysine derivatives. This leads to increase in oxidative stress which evokes the enzymatic and non-enzymatic antioxidant system against accumulation of ROS and lipid peroxidation, further preventing liver from severe dysfunction (MatÉs, Pérez-Gómez, & De Castro, 1999). Enzymatic system encompasses several enzymes like CAT and GPx (Jia et al., 2011), which catalyzes hydrogen peroxide transforming to H2O and O2. SOD is also one of the antioxidant enzymes responsible for transferring superoxide radicals to less activated compounds. We found that liver CAT and SOD activities were facilitated to maintain the balance of ROS production in TAA-treated rats. Thus, the produced ROS may have turned into nontoxic compounds. However, activities of GPx and GSH contents in livers were decreased, accompanied with higher TBARS values after a TAA treatment, implicating that overproduction of ROS could hardly be removed by those mentioned under TAA
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Fig. 2 – Effects of D-glucuronolactone (lactone) on gene expressions of KLF6, NFκB, COLα1, AP1, α-SMA, and IL6 in the livers of thioacetamide (TAA)-treated rats. The data are given as mean ± SEM. Different letters on data bars of each target gene represent significant difference (p < 0.05). The value of hepatic KLF6, NFκB, COLα1, AP1, α-SMA, and IL6 mRNA expressions were normalized to the value of GAPDH and values of rats in TAA and Lactone groups were expressed relatively to the average values for rats in the Control group, which was set to 1.0. Control: Saline (i.p) + normal distilled water (oral gavage); TAA: TAA + normal distilled water (oral gavage); Lactone: TAA+ 75 mg lactone/kg bw (oral gavage).
treatment. Similar results have been reported in other oxidative stress-induced models, and supplementation of natural ingredients, i.e. Antrodia camphorata (Chou, Chang, Hung, Chen, & Chiu, 2013) and litchi (Litchi chinensis Sonn.) flower–water extract (Chang et al., 2013) which present hepatoprotective effects via enhancing antioxidant capacities. Hence, lactone attenuated the increased oxidative stress; that is, increasing SOD and GPx activities as well as GSH contents and TEAC levels, thus decreasing TBARS values. Little evidence indicated that lactone has antioxidant capacity. Nevertheless, metabolites of lactone are able to help to excrete toxin by conjugating with xenobiotics to form more hydrophilic compounds through Phase II detoxification and further reduce the oxidative stress (Gao, Dinkova-Kostova, & Talalay, 2001). This indirectly antioxidant effect may underlie the relatively higher activities of SOD and GPx, and further provide a hepatoprotective effect. Liver fibrosis, the advanced symptom of excessive ROS targeting on liver, features accumulation of collagen secreted by HSCs, which is activated by inflammatory cells (Bataller & Brenner, 2005). The inflammatory response causes stimulation of related genes (IL6, IL-1β and TNF-α) and further neutrophil infiltration, which can be observed in our result (Fig. 3A). The transcriptional factors like KLF6, NFκB, and AP-1 are also involved in both inflammation and HSC activation. The inhibition of NFκB, an essential role in proinflammatory response activated by TNFα, has received much attention in antioxidant and anti-inflammatory effects. An induction of NFκB associated with activated HSC often relates to liver damage because it imposes a constraint on HSC apoptosis, further leading to aggregating hepatic fibrosis (Mann & Smart, 2002). In other words, those transcriptional factors and cytokines have a relatively positive correlation with liver fibrosis, and in accordance with our results indicating that liver damage had been
induced by a TAA treatment through an elevation of those factors. The activated HSCs transfer into myofibroblast-like cells, with expressions of COLα1 as well as α-SMA and trigger a generation of ECM, consisting mainly of collagen types I and III after hepatic necrosis in order to conduct wound-healing response on injured liver to attain liver regeneration (Stalnikowitz & Weissbrod, 2003). Thus, the dissemination of ECM in liver depends on where damage occurred (Bataller & Brenner, 2005) and often aggregated in portal tracts, around hepatocytes and bile ducts (Pinzani & Rombouts, 2004). Those features of liver fibrosis have prospect to be reversible (Bataller & Brenner, 2005) if continued damage is prohibited by functional ingredients. Based on histopathological findings from this study, lactone exhibited a capacity of anti-fibrosis which apparently decreased collagen accumulation (Fig. 3A) and it is probably caused by a down-regulation of COLα1. Lower expression of α-SMA in Lactone group also indicated the less severe fibrosis. The aforementioned capacity of lactone, including anti-inflammation and anti-fibrosis effects, induces a benign cycle to promote each other leading to the amelioration of liver injury. Lack of evidence in lactone supports the assumption that lactone can ameliorate liver damage; however, according to our results, liver fibrosis of rats induced by TAA can be attenuated by supplementing lactone. Hence, we assumed that those effects are attributed to lactone itself and its metabolites. In chronic liver fibrosis model built by carbon tetrachloride (CCl4) injection on mice, lactone attenuated the sharp rise of AST and ALT which demonstrated the potential hepatoprotective capacity of lactone (Chang & Chien, 2011). Furthermore, some energy drinks that contain lactone claim that together with other related derivatives especially glucaric acid they have anticancer effects via inhibition of β-glucuronidase (Higgins, Tuttle, & Higgins, 2010). The antioxidant effects may be derived from
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Fig. 3 – Effects of D-glucuronolactone (lactone) on (A) gross appearance, H&E staining, and Masson trichrome staining, (B) histology activity index score (HAI score), and (C) Metavir score of the liver section in the thioacetamide (TAA)-treated rats. CV is an abbreviation of central vein.
ascorbic acid synthesized from one of the metabolites of lactone-L-gulunolactone through gulunolactone oxidase which is deficient in humans (Marsh, 1963). Bastway Ahmed, Hasona, and Selemain (2010) also suggested that ascorbic acid had the ability to attenuate TAA-induced liver injury relying on inhibition of elevated TBARS values and relative increase of GSH contents. Another metabolite of lactone – glucuronic acid participates in Phase II detoxification of livers via a conjugation with xenobiotics to prevent liver from further damage caused by drug or other toxins. The elevated Phase II activity by glucuronic acid may recover the imbalance of Phase I and Phase II which closely relates to cancer induction (Wilkinson & Clapper, 1997). It has also been reported that the kombucha
tea containing glucuronic acid can provide hepatoprotective effects on carbon tetrachloride-induced liver injury (Murugesan et al., 2009). Those investigations support our findings of lactone as a potential functional ingredient for alleviating the development of liver fibrosis, and it may be correlated with itself or its metabolites.
5.
Conclusion
In conclusion (Fig. 4), lactone can attenuate TAA-induced liver fibrosis via two major pathways including anti-fibrosis
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Fig. 4 – Schematic representation of protective mechanism by which D-glucuronolactone (lactone) alleviates thioacetamide (TAA)-induced liver damage.
(reductions of collagen accumulation in liver and related gene expressions), and anti-inflammation (attenuations of elevated ALT, cytokines and expressions of related genes). Lactone provides great antioxidant capacity (potentiation of SOD and GPx activities) as well and further helps to prevent an advanced liver injury. This investigation implicated a novel view on function of lactone, and it could be a fundamental functional ingredient for prevention of TAA-induced liver fibrosis.
Acknowledgements We acknowledge the funding of this research by the National Science Council Taiwan (R.O.C.) (Project: NSC 101-2815-C-002152-B) and a kind offer of D-glucuronolactone (C6H8O6, lactone) from Forever Chemical Co., Ltd. (Tauyan County, Taiwan).
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