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Thymosin alpha 1 attenuates lipid peroxidation and improves fructose-induced steatohepatitis in rats
Clinical Biochemistry 38 (2005) 540 – 547
Thymosin alpha 1 attenuates lipid peroxidation and improves fructose-induced steatohepatitis in rats ¨ mer Coskunb, Ahmet Gqrela, Mehmet Kanterb, Murat Cana, Ferah Armutcua,T, O Fatma Ucarc, Murat Unalacakd a
Department of Biochemistry and Clinical Biochemistry, Zonguldak Karaelmas University, Faculty of Medicine, Zonguldak, Turkey b Department of Histology and Embryology, Zonguldak Karaelmas University, Faculty of Medicine, Turkey c Ankara Numune Education and Research Hospital, 2nd Classic of Biochemistry, Ankara, Turkey d Department of Family Medicine, Zonguldak Karaelmas University, Faculty of Medicine, Turkey Received 6 August 2004; received in revised form 6 January 2005; accepted 17 January 2005 Available online 23 February 2005
Abstract Objectives: The aim of this study was to investigate the effects of thymosin a1 (Ta1) in rats having fructose-induced steatosis. Fructose leads to experimental steatosis in the liver by exerting its effect on some components of the oxidant/antioxidant system, and on several cytokines (interleukin-1h, -2, and -6) in blood. Methods: Twenty-four rats at random were divided into three groups (each group containing eight animals); the control group (C), which received a purified diet; the high-fructose-fed group (F); and the high-fructose-fed and Ta1 injected group (F + T). After the experimental period of 10 days, liver lipid peroxidation and antioxidant status, and blood IL-1h, IL-2, and IL-6 levels were quantified. Results: In comparison with the C group, the F group had a higher nitric oxide (NO) level, xanthine oxidase (XO) activity, and lipid peroxidation, as indicated by concentrations of thiobarbituric acid reactive substances (TBARS), and lower superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in the liver. In the F + T group, these markers were near the values of the control group. In addition, increased IL-1h and IL-6 levels were kept at near to normal levels with treatment of Ta1, but not IL-2 levels. In the F group, the most consistent findings in the histologic sections of liver tissues were the macrovesicular and microvesicular steatosis. Ta1 treatment protected the majority of the liver cells, while minimal macrovesicular and microvesicular steatosis was observed in the remaining cells. Conclusions: These results show that a high-fructose diet in rats leads to hepatic steatosis and a defect in the free radical defense system, and that treatment of Ta1 may improve these biochemical and morphologic changes in the fructose-fed rat livers. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Fructose-induced steatosis; Oxidative stress; Nitric oxide; Cytokines; Thymosin a1; Rats
Introduction Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are the two most common chronic liver diseases in the general population of the United States, with prevalences of 20% and 3%, respectively. NAFLD includes a wide spectrum of liver injury ranging from simple steatosis to steatohepatitis, fibrosis, and cirrhosis. Steatosis, which is characterized by intracellular acT Corresponding author. Fax: +90 372 2610155. E-mail address: [email protected] (F. Armutcu).
cumulation of lipids in cytoplasmic vacuoles, represents the most common alteration found in the liver in the general population. It appears to be associated with oxidative events regardless of the specific cause [1,2]. Evidence of an increased generation of reactive oxygen species (ROS) has been described in several animal models of fatty liver, including alcohol or caffeine administration; however, within a few days, the choline-deprived diet produces massive liver steatosis, predominantly macrovesicular, without evidence of inflammation and/or fibrosis [3–5]. Environmental factors, such as diets and toxins, can also deteriorate hepatic fatty acid synthesis and oxidation. Dietary fructose hepatic lipogenesis
0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.01.013
F. Armutcu et al. / Clinical Biochemistry 38 (2005) 540–547
increases and exerts acute effects on hepatocyte energy homeostasis. When adult Wistar rats are given 10% fructose in the drinking water for 48 h, hepatic fatty acid synthase is induced and de novo fatty acid synthesis and esterification increase significantly [6–8]. The underlying mechanisms for the detrimental consequences of a high-fructose diet in animal models are not clear. However, the ability of fructose to induce peroxidation of membrane lipids is widely reviewed in the literature [9,10]. In addition, lipid peroxidation is associated with inflammation and cytokine activation, thus increased ROS could cause more lipid peroxidation and induction of proinflammatory cytokines in hepatocytes [11,12]. No therapy for NASH has been proven to be clearly effective. Currently, treatment of hepatic steatosis is focused on modifying risk factors such as obesity, diabetes mellitus, and hyperlipidemia. Thymosin alpha 1 (Ta1) is a hormone produced by thymic stromal cells that can augment host defense mechanisms by increasing the production of T cells by the thymus. It has been used in medicine for diagnosis and treatment of many diseases [13,14]. Moreover, it has been shown to have immunomodulatory [15], antitumor [16], antioxidant [17], and wound healing [18] properties. The potential therapeutic efficacy of antioxidants in steatohepatitis is not yet clear. The aim of this study was to evaluate possible antioxidant and protective roles of Ta1 in prevention of fructose-induced hepatic steatosis.
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anesthetized with 0.5 mg/kg of ketamin (KetalarR, EczacVbaYV Farma) intraperitoneally. Blood from the heart was collected after entering the abdominal and thoracic cavities into tubes containing potassium EDTA using 10-mL syringes at the time of death. Blood samples were centrifuged at 1000 g for 10 min at 48C to remove plasma. Aliquots of the samples were transferred into polyethylene tubes to be used in the assay of biochemical parameters and were stored at 708C until analysis. Approximately 200 mg of liver tissue from each rat was kept on ice for the lipid peroxidation and the other biochemical assays. All tissues were washed two times with cold saline solution, placed into glass bottles, labeled, and stored in a deep freezer ( 408C) until processing (maximum 10 h). Liver tissues were homogenized in four volumes of ice-cold Tris–HCl buffer (50 mM, pH 7.4) using a glass teflon homogenizer (Ultra Turrax IKA T18 Basic) after cutting the tissues into small pieces with scissors (for 2 min at 5000 rpm). Nitric oxide, TBARS, xanthine oxidase (XO), and liver triglyceride measurements were made at this stage. The homogenate was then centrifuged at 5000 g for 60 min to remove debris. The clear upper supernatant fluid was taken and GSH-Px activities and protein concentration were carried out at this stage. The supernatant solution was extracted with an equal volume of an ethanol/chloroform mixture (5/3; v/v). After centrifugation at 5000 g for 30 min, the clear upper layer (the ethanol phase) was taken and used in the SOD activity and protein assays. All preparation procedures were performed at +48C.
Materials and methods Biochemical parameters Animals and experimental design Male Wistar albino rats weighting 280–300 g were used in this study. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animals were housed at 20–248C with a 12-h light, 12-h dark cycle and supplied with standard rat chow freely available. Rats were randomly assigned to one of the three groups (each group containing eight animals); the control group (C), which received a purified diet; the highfructose-fed group (F); and high-fructose-fed plus Ta1 injected group (F + T). Fructose (Merck Darmstadt Germany) dissolved in tap water 10% (w/v) was administrated to their drinking waters ad libitum. Thymosin a1 (Sigma; St Louis, MO, USA) was dissolved in 5 mL sterile saline and was injected into the peritoneum at a dose of 10 Ag/kg every other day, and a sterile saline solution was also injected in the same manner to the control and high-fructose-fed groups. The first dose of Ta1 was given 2 days before the high-fructose diet. The rats were sacrificed 10 days after Ta1 injection. Sample preparation The animals were starved overnight for 12 h before the blood was collected. At the time of death, rats were
Liver thiobarbituric acid reactive substances (TBARS) TBARS are products of the oxidative degradation of polyunsaturated fatty acids, in particular, malondialdehyde (MDA). We used the method of Draper and Hadley [19]. This method is based on the fact that lipid peroxides and thiobarbituric acid (TBA) react to form a pink pigment with a maximum absorption at 532 nm. For this purpose, 2.5 mL of 100 g/L trichloroacetic acid (TCA) solution was added to 0.5 mL erythrocytes in each centrifuge tube and placed in a boiling water bath for 15 min. After cooling in tap water, the mixture was centrifuged at 1000 g for 10 min, and 2 mL of the supernatant was added to 1 mL of 0.67% (w/v) TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled in tap water and its absorbance was measured using a spectrophotometer at 532 nm (ShVmadzu UV-1601, Japan). The concentration of TBARS was calculated by the absorbance coefficient of the MDA–TBA complex, 1.56 105 cm 1 M 1, and was expressed in Amol/g protein. TBA and TCA used in this assay procedure were obtained from Merck (Darmstadt Germany). Liver superoxide dismutase activity Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al. [20]. The principle of the method is based on the inhibition of nitroblue
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tetrazolium (NBT) reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the liver homogenate after a 1.0-mL ethanol/chloroform mixture (5/3, v/v) was added to the same volume of the hemolysate and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. SOD activity was also expressed as units per gram of protein. All chemicals used in this assay procedure (xanthine, xanthine oxidase, NBT) were obtained from Sigma (St, Louis, MO, USA), and CuCl2, bovine serum albumin, EDTA, Na2CO3, (NH4)2SO4, chloroform, and ethanol from Merck (Darmstadt Germany). Liver glutathione peroxidase activity Glutathione peroxidase (GSH-Px, EC 1.6.4.2) activity was measured by the method of Paglia and Valentine [21]. The enzymatic reaction in the tube, which contains the following items: NADPH, reduced glutathione (GSH), sodium azide, and glutathione reductase, was initiated by the addition of H2O2 and the change in absorbance at 340 nm was monitored by a spectrophotometer. This assay used commercial chemicals supplied by Sigma (St. Louis, MO, USA). Liver nitric oxide levels As nitric oxide measurement is very difficult in biological specimens, erythrocyte nitrite (NO2 ) and nitrate (NO3 ) were estimated as an index of NO production. Quantitation of nitrate and nitrite was based on the Griess reaction in which a chromophore with a strong absorbance at 540 nm is formed by reaction of nitrite with a mixture of naphthlethylendiamine and sulphanilamide [22]. Samples were initially deproteinized with Somogyi reagent [23]. Following cleanup, an aliquot of the sample was mixed with fresh reagent and the absorbance was measured in a spectrophotometer (ShVmadzu UV-1601, Japan) to give the nitrite concentration. For nitrate detection, a second aliquot was treated with copporized cadmium in glycine buffer at pH 9.7 (2.5 to 3 g of Cd granules for a mL reaction mixture) to reduce nitrate to nitrite. The concentration of nitrite in this aliquot thus represented the total nitrate plus nitrite. The nitrate concentration in the sample was thus given by the difference between the two aliquots. A standard curve was established with a set of serial dilutions of sodium nitrite (10 8 to 10 3 mol/L). Linear regression was done using the peak areas from the nitrate and nitrite standards. The resulting equation was then used to calculate the unknown sample concentrations. All chemicals used in this assay procedure were obtained from Sigma except cadmium granules (Merck). Results were expressed as Amol/g protein. Protein assays were made by the Lowry et al. method [24]. All samples were assayed in duplicate. For the measurement of liver triglyceride levels, liver tissue was extracted with methanol/chloroform (1:2) according to the method of Folch et al. [25]. Triglyceride levels in the liver extract and plasma were determined using colorimetry kits (Triglyceride,
CHOD-PAP) in analyzer (Cobas Integra 800, Roche Diagnostics USA). Plasma levels of IL-1h, IL-2, and IL-6 levels were measured with ELISA kits using monoclonal antibodies specific to human IL-1h, IL-2, and IL-6 (BioSource International Inc., California, USA). According to the manufacturer’s data, the lower limits of detection of IL-1h, IL-2, and IL-6 with these assay systems were b3 pg/ mL, b5 pg/mL, and b8 pg/mL, respectively. Histopathological procedure Liver tissues were harvested from the sacrificed animals, and the fragments from tissues were fixed in 10% neutral formalin solution, embedded in paraffin and then stained with hematoxylin and eosin. Preparations were evaluated by a bright field microscope (Olympus BX51, Tokyo, Japan) and were photographed by a Spot Insight QE (Diagnostic Instruments, Silver Spring, USA) camera. Image analysis The system used is composed of a PC, hardware, and software (Image-Pro Plus 5.0-Media Cybernetics, Silver Spring, USA) for image acquisition and analysis and a Spot Insight QE (Diagnostic instruments, Silver Spring, USA) camera and optical microscope. The method requires preliminary software procedures of spatial calibration (micron scale) and setting of color segmentation for quantitative color analysis. Fifty liver preparations from each group were chosen randomly. The area of macrovesicular steatosis in the livers was measured. The percentage of the macrovesicular steatosis was calculated according to these results. The investigator who performed these measurements was unaware of the experiment. For the image analysis of the macrovesicular steatosis data, a nonparametric test (Kruskal–Wallis) was used. Differences were considered to be statistically significant if P b 0.05. Statistical analysis Data were analyzed by SPSS for Windows. Distribution of groups was analyzed with a one-way Kolmogrov– Smirnov test. All groups showed normal distribution, so that parametric statistical methods were used. A one-way ANOVA test was performed and post hoc multiple comparison were done with LSD. Results were presented as mean F standard deviation (SD) and a probability value of less than 0.05 was considered statistically significant.
Results Biochemical findings All the biochemical data are summarized in Tables 1 and 2. As shown in Table 2, the high-fructose diet induced
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Table 1 The liver tissue TBARS and NO levels, the activities of XO, SOD, and GSH-Px in control, fructose-induced, and fructose-induced plus thymosin a1-treated groups C TBARS (nmol/g protein) NO (Amol/g protein) XO (U/g protein) SOD (U/g protein) GSH-Px (U/g protein)
F
F+T a
29.5 F 2.36
42.1 F 4.26
0.19 6.6 70.0 31.5
0.26 F 11.1 F 51.3 F 27.6 F
F F F F
0.03 1.08 8.92 4.20
0.02a 2.20a 8.44a 3.24
33.6 F 1.11b,c 0.20 8.7 63.2 29.5
F F F F
0.02b 0.90c,d 5.46d 3.56
Values are means F SD for 8 rats. a P b 0.001 when compared with group C. b P b 0.001 when compared with group F. c P b 0.05 compared with group C. d P b 0.01 when compared with group F. Fig. 1. Liver histology in control rats (H & E; scale bar = 25 Am).
remarkable hypertriglyceridemia in both liver and plasma in our experimental model. Fructose-induced steatosis was also confirmed histologically (Fig. 2). In the F group, the levels of triglyceride in the plasma and liver were significantly increased compared to the control group (P b 0.001). Increase in plasma and liver triglyceride levels was observed to be less in the Ta1 injected group compared to the control group (P b 0.01). In the F group, the levels of TBARS in the liver were significantly increased compared to control rats (P b 0.001). In the F + T group, TBARS levels were significantly lower compared to the F group (P b 0.001) in the liver. In the F group, the level of NO in the liver was significantly increased compared to control rats (P b 0.001). In the F + T group, NO levels were significantly lower compared to the F group (P b 0.001). When the Ta1injected group was compared with the control group, NO and SOD values did not differ significantly, but TBARS and XO levels showed significant differences (P b 0.05). There were significant changes in most of the liver tissue enzymes, except GSH-Px. The oxidant enzyme XO activity increased significantly (P b 0.001) in the F group, whereas it was significantly lower in the F + T group compared to the F group (P b 0.001). The activity of SOD was significantly increased (P b 0.001) in the F group compared to controls and significantly lower (P b 0.01) in the F + T group compared to the F group (Table 1).
As shown in Table 2, the plasma IL-1h and IL-6 levels were significantly higher (P b 0.01 and P b 0.001, respectively) in the F group compared to control rats, and the levels of these cytokines were significantly lower (P b 0.001) in the F + T group compared to the F group. Although IL-1h and IL-6 levels decreased by injection of Ta1, they did not decrease to control levels (P b 0.01). The plasma IL-2 levels were also higher (P b 0.001) in the F group compared to control rats. IL-2 levels in the F + T group were higher than the other two groups. Histopathological findings The histology of the liver was normal in the control group (Fig. 1). In the F group, the most consistent findings in the histologic sections of liver tissues stained with hematoxylin and eosin were the macrovesicular and microvesicular steatosis in the liver tissues (Fig. 2). Ta1 treatment protected the majority of the liver cells. Nevertheless, minimal macrovesicular and microvesicular steatosis was observed in the remaining cells. In addition, a few cells were
Table 2 The plasma IL 1h, IL-2, and IL-6 levels in control, fructose-induced, and fructose-induced plus thymosin a1-treated groups Parameters Trigliserid (mg/dL) Trigliserid (mg/dL wet tissue) IL 1h (pg/mL) IL 2 (pg/mL) IL 6 (pg/mL)
C
F
F+T a
44.1 F 5.19 346.4 F 36.8
68.0 F 12.60 808.2 F 110.8a
21.4 F 5.82 15.7 F 3.49 9.4 F 1.15
41.5 F 3.97a 25.9 F 2.62a 19.6 F 2.01a
Values are means F SD for 8 rats. a P b 0.001 when compared with group C. b P b 0.001 when compared with group F. c P b 0.01 when compared with group C. d P b 0.01 when compared with group F.
46.8 F 5.46b 542.4 F 46.4b,c 33.9 F 4.11c,d 34.6 F 6.24b,c 14.5 F 2.70b,c
Fig. 2. After 10 days of fructose treatment, hepatocellular degeneration, macrovesicular (arrow) and microvesicular steatosis were observed in the hepatic lobule, in absence of any inflammation (H & E; scale bar = 25 Am).
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Fig. 3. Liver histology of thymosin-treated rats. Minimal degeneration of hepatocytes and microvesicular steatosis are seen. However, some macrovesicular steatosis (arrow) are still observed (H & E; scale bar = 25 Am).
indicated to have picnotic nucleus (Fig. 3). As shown in Table 3, Ta1 treatment protected the intensity of steatosis in the liver significantly in comparison with untreated highfructose-fed animals (P b 0.05). The area of macrovesicular steatosis was measured in the livers of the F and F + T groups. The percentage of the macrovesicular steatosis was calculated according to the results shown in Table 3. Fructose induction resulted in a significant increase of the area of macrovesicular steatosis. Ta1 treatment prevented macrovesicular steatosis in liver.
Discussion Nonalcoholic steatohepatitis (NASH) is one of the most common liver diseases encountered in the United States and Europe. Although this term refers to a spectrum of hepatic pathology that resembles alcoholic liver disease, it refers to a wide spectrum of liver damage, ranging from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis [2]. A dtwo-hitT concept of disease pathogenesis has been proposed. The first hit is steatosis, and this is postulated to sensitize the liver to the second hit, which may be oxidative stress or abnormal cytokine production. Oxidative stress and lipid peroxidation are candidates for the second hit in the pathogenesis of NASH. Both animal data and human studies have shown a link between NASH and oxidative stress and lipid peroxidation [26,27]. The underlying mechanisms for the detrimental consequences of a high-fructose diet in animal models are not clear. It is known that hepatic fatty acid synthase is induced and de novo fatty acid synthesis is increased in Wistar rats that have been fed fructose in drinking water [7,8]. It is now established that oxidative damage is present in several animal models of steatohepatitis. Oliveira et al. have found that oxidative stress increased in the experimental hepatic steatosis caused by a choline-deficient diet [5]. Fructose-rich diets have been shown to have deleterious metabolic effects, including
glucose intolerance, insulin resistance, dyslipidemia, and liver dysfunction. Some of these metabolic effects of fructose are attributed to its rapid hepatic uptake and the fact that it bypasses the phosphofructokinase regulatory step in glycolysis [28,29]. Fructose-induced increase in triacylglycerols may be due to stimulation of triacylglycerol synthesis. Moreover, the possibility exists that fructose feeding facilitates oxidative damage. Following fructose consumption, excesses of NADH and NADPH are known to occur during the metabolism of fructose via the pentose shunt and sorbitol pathway [30,31]. In addition, fructose feeding results in increased xanthine oxidase activity, and to glyceraldehyde production, which can induce free radicals. In the metabolism of hypoxanthine by the enzyme xanthine oxidase, both the superoxide radical and hydrogen peroxide can be generated [32,33]. Indeed, this study shows that fructose-fed rats were more susceptible to peroxidative damage as measured by thiobarbituric acid reactive species. Fructose feeding results in increased liver xanthine oxidase activity and nitric oxide production, which can induce ROS. NO may potentiate cytotoxicity by reaction with superoxide anion to form peroxynitrite, which then causes protein nitration and tissue injury. The finding that intrahepatic accumulation of nitrotyrosine is associated with the histological severity of NASH strongly suggests that oxidative injury may play a significant role in the pathogenesis of NASH [34,35]. However, in the presence of massive injury, greatly increased NO production might induce the hepatocytes to progress to irreversible channel necrosis and cell death [36]. On the other hand, Zhu and Fung [37] reported that NO protects against liver injury by scavenging lipid radicals and inhibiting the lipid peroxidation chain reaction. Inducible NO may initiate and play a key role in the metabolic and functional stress responses of hepatocytes against endotoxin and cytokines. Sass et al. [38] found that iNOS-derived NO regulates proinflammatory genes in vivo, contributing to inflammatory liver injury. Steatosis-induced lipid peroxidation and ROS can consume antioxidant enzymes and vitamins [12]. Depletion of these protective substances may hamper ROS inactivation and increase lipid peroxidation and ROS-mediated damage. Our results also show that, in fructose-fed rats, the balance between prooxidants and antioxidants is disturbed by the increased activities of antioxidant enzymes such as SOD and GSH-Px.
Table 3 Comparison area of the macrovesicular steatosis in the liver A (control), B (high-fructose-fed), and C (Ta1 treatment) groups C Mean area of macrovesicular steatosis in livers
0%
F
F+T a
12.36 F 1.23%
4.34 F 0.28%b,c
The Kruskal–Wallis test was used for statistical analysis. Values are expressed as mean F SD, and n = 50 liver preparations for all groups. a P b 0.001 when compared with group C. b P b 0.001 when compared with group F. c P b 0.01 when compared with group C.
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This finding may indicate that the hepatic antioxidant enzymatic defense system is impaired in NASH. The balance between oxidative stress and antioxidant defense mechanisms may be impaired by depletion of enzymatic antioxidants and increased plasma levels of TBARS and NO in patients with NASH [39]. Several experimental therapies, including treatment with bile acids, antibiotics, nutritional supplements, and antioxidants, have had anecdotal success in selected patients, but improved understanding of the pathogenesis and natural history of NASH will be required to develop generally effective therapy for the disorder [2,40]. The release of ROS is thought to be responsible for the general liver damage that occurs, as evidenced by the ability to attenuate hepatotoxicity by antioxidant treatment or cytokine neutralization [41]. In primary steatohepatitis, this may be due to several vicious cycles involving steatosis, lipid peroxidation, mitochondrial damage, enchanced ROS formation, increased TNF-a, and depletion of antioxidants. TNF-a impairs mitochondrial respiration and also causes opening of the mitochondrial permeability transition pore, which may further increase mitochondrial ROS formation and lipid peroxidation [12,39]. The release of TNF-a activates specific redox sensitive kinases that can upregulate proinflammatory pathways and enhance insulin resistance [42]. Finally, ROS-associated lipid peroxidation and cytokines may be involved in the inflammatory cell infiltrate, thus extensive ROS formation in secondary NASH could play a role in necroinflammation [11]. Interleukins are regulatory peptides that can be produced by virtually every nucleated cell in the body, including liver cells [43]. Most types of cells in the liver, including Kupffer cells, hepatocytes, and stellate cells, either synthesize or respond to cytokines, whereas proinflammatory cytokines such as TNF-a, interleukin-6, and interleukin-1h are mainly involved in cholestasis and the synthesis of acute-phase proteins by activated Kupffer cells and hepatocytes [44]. Altered Kupffer cell production of various cytokines is also likely to deplete the liver of certain populations of lymphocytes, thereby changing vulnerability to the hepatotoxic actions of agents that activate both innate and T-cellmediated immune responses [45]. Abnormal cytokine production in NASH patients may also be due to abnormal macrophage function. TNF-a initiates various intracellular signals that increase mitochondrial permeability and the release of reactive oxygen species [11]. Mitochondrial ROS formation is further increased, which could cause more lipid peroxidation, cytokine induction, and fibrogenesis [12]. Faure et al. reported that a high-fructose diet in rats leads to insulin resistance and a defect in the free radical defense system, increasing oxidative stress. According to their study with vitamin E supplementation, there was improvement in insulin sensitivity in fructose-fed rats [9]. It is clear that the oxidant/antioxidant balance plays an important role in regulation of cytokines. Increased levels of proinflammatory cytokines are found in both the plasma and liver in alcoholic
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hepatitis. The plasma IL-6 concentration appears to correlate well with the clinical severity and course of alcoholic hepatitis. Inflammatory cytokines, including IL-1 and TNFa, are thought to associate with the pathophysiology of liver disease [11,46]. IL-1 and IL-6 are responsible for many cellular events that are present in acute inflammation. These cytokines increase acute phase protein synthesis in hepatocytes. IL-1 may induce the formation of oxygen free radicals, which mediate the neutrophil–endothelium interaction causing cell dysfunction and destruction [47,48]. Antioxidants such as vitamin E, N-acetylcysteine, betaine, and others may be beneficial in the treatment of NASH [49]. Abdelmalek et al. reported that betaine (a metabolite of choline and antioxidant) treatment provides histological improvement in NASH [50]. Ta1 is a synthetically produced thymic peptide that has been shown to enhance the response of T cells and stimulate the production of endogenous interleukins and interferons. Ta1 promotes angiogenesis and wound healing [14,18]. It is able to enhance natural killer cell activity and it has been successfully tested in patients with chronic hepatitis B. Moreover, it has been proposed that Ta1 may modulate the action of IL-2 [51,52]. Parallel to this information, IL-2 levels were also shown in our study to be increased remarkably in the Ta1-treated group. The thymic hormones were reported to be effective on lipid peroxidation. AdemoWlu et al. have reported that plasma and erythrocyte TBARS levels significantly decreased in the Ta1-injected rats [53]. Tada et al. showed that thymic hormones increased GSH levels in tacrolimus-induced nephrotoxicity [54]. Our data indicate that Ta1-treated rats developed significantly less liver steatosis and lipid peroxidation. Oxidative damage is also prominent in the liver of humans with NASH, as well as in the histologically similar disorder of alcoholic hepatitis [11,55]. Histopathological examinations also demonstrated that Ta1 also effectively stabilizes the severity of hepatic lesions in the liver of fructose-induced rats. Triglycerides are the main component of accumulated fatty droplets. In the present study, both liver and fasting serum triacylglycerol levels were significantly elevated by dietary treatment. However, serum ALT levels also were not significantly increased in this study. Stravitz and Sanyal reported that a low normal ALT value does not guarantee freedom from underlying steatohepatitis with advanced fibrosis [56]. In conclusion, these data provide experimental evidence for a promising antioxidant and protective effect of Ta1 in fructose-induced steatosis. Taken together, the data presented here indicate that: (a) ROS may be involved in the mechanism of nonalcoholic steatohepatitis; (b) the generation of ROS not only induces lipid peroxidation, but also modulates the production of cytokines (especially proinflammatory); (c) lipid peroxidation and cytokines may play a role in fructose-induced hepatocellular injury and thymosin a1 may have some beneficial effects on the treatment of steatosis by normalizing blood cytokine levels and by substantially protecting liver cells against free radical injury.
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Altogether, future investigations are necessary to elucidate the role of thymosin a1 in NASH prevention, and further studies at different doses of thymosin a1 are needed to determine the most appropriate dosage.
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Thymosin alpha 1 attenuates lipid peroxidation and improves fructose-induced steatohepatitis in rats ¨ mer Coskunb, Ahmet Gqrela, Mehmet Kanterb, Murat Cana, Ferah Armutcua,T, O Fatma Ucarc, Murat Unalacakd a
Department of Biochemistry and Clinical Biochemistry, Zonguldak Karaelmas University, Faculty of Medicine, Zonguldak, Turkey b Department of Histology and Embryology, Zonguldak Karaelmas University, Faculty of Medicine, Turkey c Ankara Numune Education and Research Hospital, 2nd Classic of Biochemistry, Ankara, Turkey d Department of Family Medicine, Zonguldak Karaelmas University, Faculty of Medicine, Turkey Received 6 August 2004; received in revised form 6 January 2005; accepted 17 January 2005 Available online 23 February 2005
Abstract Objectives: The aim of this study was to investigate the effects of thymosin a1 (Ta1) in rats having fructose-induced steatosis. Fructose leads to experimental steatosis in the liver by exerting its effect on some components of the oxidant/antioxidant system, and on several cytokines (interleukin-1h, -2, and -6) in blood. Methods: Twenty-four rats at random were divided into three groups (each group containing eight animals); the control group (C), which received a purified diet; the high-fructose-fed group (F); and the high-fructose-fed and Ta1 injected group (F + T). After the experimental period of 10 days, liver lipid peroxidation and antioxidant status, and blood IL-1h, IL-2, and IL-6 levels were quantified. Results: In comparison with the C group, the F group had a higher nitric oxide (NO) level, xanthine oxidase (XO) activity, and lipid peroxidation, as indicated by concentrations of thiobarbituric acid reactive substances (TBARS), and lower superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities in the liver. In the F + T group, these markers were near the values of the control group. In addition, increased IL-1h and IL-6 levels were kept at near to normal levels with treatment of Ta1, but not IL-2 levels. In the F group, the most consistent findings in the histologic sections of liver tissues were the macrovesicular and microvesicular steatosis. Ta1 treatment protected the majority of the liver cells, while minimal macrovesicular and microvesicular steatosis was observed in the remaining cells. Conclusions: These results show that a high-fructose diet in rats leads to hepatic steatosis and a defect in the free radical defense system, and that treatment of Ta1 may improve these biochemical and morphologic changes in the fructose-fed rat livers. D 2005 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Fructose-induced steatosis; Oxidative stress; Nitric oxide; Cytokines; Thymosin a1; Rats
Introduction Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are the two most common chronic liver diseases in the general population of the United States, with prevalences of 20% and 3%, respectively. NAFLD includes a wide spectrum of liver injury ranging from simple steatosis to steatohepatitis, fibrosis, and cirrhosis. Steatosis, which is characterized by intracellular acT Corresponding author. Fax: +90 372 2610155. E-mail address: [email protected] (F. Armutcu).
cumulation of lipids in cytoplasmic vacuoles, represents the most common alteration found in the liver in the general population. It appears to be associated with oxidative events regardless of the specific cause [1,2]. Evidence of an increased generation of reactive oxygen species (ROS) has been described in several animal models of fatty liver, including alcohol or caffeine administration; however, within a few days, the choline-deprived diet produces massive liver steatosis, predominantly macrovesicular, without evidence of inflammation and/or fibrosis [3–5]. Environmental factors, such as diets and toxins, can also deteriorate hepatic fatty acid synthesis and oxidation. Dietary fructose hepatic lipogenesis
0009-9120/$ - see front matter D 2005 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2005.01.013
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increases and exerts acute effects on hepatocyte energy homeostasis. When adult Wistar rats are given 10% fructose in the drinking water for 48 h, hepatic fatty acid synthase is induced and de novo fatty acid synthesis and esterification increase significantly [6–8]. The underlying mechanisms for the detrimental consequences of a high-fructose diet in animal models are not clear. However, the ability of fructose to induce peroxidation of membrane lipids is widely reviewed in the literature [9,10]. In addition, lipid peroxidation is associated with inflammation and cytokine activation, thus increased ROS could cause more lipid peroxidation and induction of proinflammatory cytokines in hepatocytes [11,12]. No therapy for NASH has been proven to be clearly effective. Currently, treatment of hepatic steatosis is focused on modifying risk factors such as obesity, diabetes mellitus, and hyperlipidemia. Thymosin alpha 1 (Ta1) is a hormone produced by thymic stromal cells that can augment host defense mechanisms by increasing the production of T cells by the thymus. It has been used in medicine for diagnosis and treatment of many diseases [13,14]. Moreover, it has been shown to have immunomodulatory [15], antitumor [16], antioxidant [17], and wound healing [18] properties. The potential therapeutic efficacy of antioxidants in steatohepatitis is not yet clear. The aim of this study was to evaluate possible antioxidant and protective roles of Ta1 in prevention of fructose-induced hepatic steatosis.
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anesthetized with 0.5 mg/kg of ketamin (KetalarR, EczacVbaYV Farma) intraperitoneally. Blood from the heart was collected after entering the abdominal and thoracic cavities into tubes containing potassium EDTA using 10-mL syringes at the time of death. Blood samples were centrifuged at 1000 g for 10 min at 48C to remove plasma. Aliquots of the samples were transferred into polyethylene tubes to be used in the assay of biochemical parameters and were stored at 708C until analysis. Approximately 200 mg of liver tissue from each rat was kept on ice for the lipid peroxidation and the other biochemical assays. All tissues were washed two times with cold saline solution, placed into glass bottles, labeled, and stored in a deep freezer ( 408C) until processing (maximum 10 h). Liver tissues were homogenized in four volumes of ice-cold Tris–HCl buffer (50 mM, pH 7.4) using a glass teflon homogenizer (Ultra Turrax IKA T18 Basic) after cutting the tissues into small pieces with scissors (for 2 min at 5000 rpm). Nitric oxide, TBARS, xanthine oxidase (XO), and liver triglyceride measurements were made at this stage. The homogenate was then centrifuged at 5000 g for 60 min to remove debris. The clear upper supernatant fluid was taken and GSH-Px activities and protein concentration were carried out at this stage. The supernatant solution was extracted with an equal volume of an ethanol/chloroform mixture (5/3; v/v). After centrifugation at 5000 g for 30 min, the clear upper layer (the ethanol phase) was taken and used in the SOD activity and protein assays. All preparation procedures were performed at +48C.
Materials and methods Biochemical parameters Animals and experimental design Male Wistar albino rats weighting 280–300 g were used in this study. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animals were housed at 20–248C with a 12-h light, 12-h dark cycle and supplied with standard rat chow freely available. Rats were randomly assigned to one of the three groups (each group containing eight animals); the control group (C), which received a purified diet; the highfructose-fed group (F); and high-fructose-fed plus Ta1 injected group (F + T). Fructose (Merck Darmstadt Germany) dissolved in tap water 10% (w/v) was administrated to their drinking waters ad libitum. Thymosin a1 (Sigma; St Louis, MO, USA) was dissolved in 5 mL sterile saline and was injected into the peritoneum at a dose of 10 Ag/kg every other day, and a sterile saline solution was also injected in the same manner to the control and high-fructose-fed groups. The first dose of Ta1 was given 2 days before the high-fructose diet. The rats were sacrificed 10 days after Ta1 injection. Sample preparation The animals were starved overnight for 12 h before the blood was collected. At the time of death, rats were
Liver thiobarbituric acid reactive substances (TBARS) TBARS are products of the oxidative degradation of polyunsaturated fatty acids, in particular, malondialdehyde (MDA). We used the method of Draper and Hadley [19]. This method is based on the fact that lipid peroxides and thiobarbituric acid (TBA) react to form a pink pigment with a maximum absorption at 532 nm. For this purpose, 2.5 mL of 100 g/L trichloroacetic acid (TCA) solution was added to 0.5 mL erythrocytes in each centrifuge tube and placed in a boiling water bath for 15 min. After cooling in tap water, the mixture was centrifuged at 1000 g for 10 min, and 2 mL of the supernatant was added to 1 mL of 0.67% (w/v) TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled in tap water and its absorbance was measured using a spectrophotometer at 532 nm (ShVmadzu UV-1601, Japan). The concentration of TBARS was calculated by the absorbance coefficient of the MDA–TBA complex, 1.56 105 cm 1 M 1, and was expressed in Amol/g protein. TBA and TCA used in this assay procedure were obtained from Merck (Darmstadt Germany). Liver superoxide dismutase activity Total (Cu–Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al. [20]. The principle of the method is based on the inhibition of nitroblue
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tetrazolium (NBT) reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was assessed in the ethanol phase of the liver homogenate after a 1.0-mL ethanol/chloroform mixture (5/3, v/v) was added to the same volume of the hemolysate and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. SOD activity was also expressed as units per gram of protein. All chemicals used in this assay procedure (xanthine, xanthine oxidase, NBT) were obtained from Sigma (St, Louis, MO, USA), and CuCl2, bovine serum albumin, EDTA, Na2CO3, (NH4)2SO4, chloroform, and ethanol from Merck (Darmstadt Germany). Liver glutathione peroxidase activity Glutathione peroxidase (GSH-Px, EC 1.6.4.2) activity was measured by the method of Paglia and Valentine [21]. The enzymatic reaction in the tube, which contains the following items: NADPH, reduced glutathione (GSH), sodium azide, and glutathione reductase, was initiated by the addition of H2O2 and the change in absorbance at 340 nm was monitored by a spectrophotometer. This assay used commercial chemicals supplied by Sigma (St. Louis, MO, USA). Liver nitric oxide levels As nitric oxide measurement is very difficult in biological specimens, erythrocyte nitrite (NO2 ) and nitrate (NO3 ) were estimated as an index of NO production. Quantitation of nitrate and nitrite was based on the Griess reaction in which a chromophore with a strong absorbance at 540 nm is formed by reaction of nitrite with a mixture of naphthlethylendiamine and sulphanilamide [22]. Samples were initially deproteinized with Somogyi reagent [23]. Following cleanup, an aliquot of the sample was mixed with fresh reagent and the absorbance was measured in a spectrophotometer (ShVmadzu UV-1601, Japan) to give the nitrite concentration. For nitrate detection, a second aliquot was treated with copporized cadmium in glycine buffer at pH 9.7 (2.5 to 3 g of Cd granules for a mL reaction mixture) to reduce nitrate to nitrite. The concentration of nitrite in this aliquot thus represented the total nitrate plus nitrite. The nitrate concentration in the sample was thus given by the difference between the two aliquots. A standard curve was established with a set of serial dilutions of sodium nitrite (10 8 to 10 3 mol/L). Linear regression was done using the peak areas from the nitrate and nitrite standards. The resulting equation was then used to calculate the unknown sample concentrations. All chemicals used in this assay procedure were obtained from Sigma except cadmium granules (Merck). Results were expressed as Amol/g protein. Protein assays were made by the Lowry et al. method [24]. All samples were assayed in duplicate. For the measurement of liver triglyceride levels, liver tissue was extracted with methanol/chloroform (1:2) according to the method of Folch et al. [25]. Triglyceride levels in the liver extract and plasma were determined using colorimetry kits (Triglyceride,
CHOD-PAP) in analyzer (Cobas Integra 800, Roche Diagnostics USA). Plasma levels of IL-1h, IL-2, and IL-6 levels were measured with ELISA kits using monoclonal antibodies specific to human IL-1h, IL-2, and IL-6 (BioSource International Inc., California, USA). According to the manufacturer’s data, the lower limits of detection of IL-1h, IL-2, and IL-6 with these assay systems were b3 pg/ mL, b5 pg/mL, and b8 pg/mL, respectively. Histopathological procedure Liver tissues were harvested from the sacrificed animals, and the fragments from tissues were fixed in 10% neutral formalin solution, embedded in paraffin and then stained with hematoxylin and eosin. Preparations were evaluated by a bright field microscope (Olympus BX51, Tokyo, Japan) and were photographed by a Spot Insight QE (Diagnostic Instruments, Silver Spring, USA) camera. Image analysis The system used is composed of a PC, hardware, and software (Image-Pro Plus 5.0-Media Cybernetics, Silver Spring, USA) for image acquisition and analysis and a Spot Insight QE (Diagnostic instruments, Silver Spring, USA) camera and optical microscope. The method requires preliminary software procedures of spatial calibration (micron scale) and setting of color segmentation for quantitative color analysis. Fifty liver preparations from each group were chosen randomly. The area of macrovesicular steatosis in the livers was measured. The percentage of the macrovesicular steatosis was calculated according to these results. The investigator who performed these measurements was unaware of the experiment. For the image analysis of the macrovesicular steatosis data, a nonparametric test (Kruskal–Wallis) was used. Differences were considered to be statistically significant if P b 0.05. Statistical analysis Data were analyzed by SPSS for Windows. Distribution of groups was analyzed with a one-way Kolmogrov– Smirnov test. All groups showed normal distribution, so that parametric statistical methods were used. A one-way ANOVA test was performed and post hoc multiple comparison were done with LSD. Results were presented as mean F standard deviation (SD) and a probability value of less than 0.05 was considered statistically significant.
Results Biochemical findings All the biochemical data are summarized in Tables 1 and 2. As shown in Table 2, the high-fructose diet induced
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Table 1 The liver tissue TBARS and NO levels, the activities of XO, SOD, and GSH-Px in control, fructose-induced, and fructose-induced plus thymosin a1-treated groups C TBARS (nmol/g protein) NO (Amol/g protein) XO (U/g protein) SOD (U/g protein) GSH-Px (U/g protein)
F
F+T a
29.5 F 2.36
42.1 F 4.26
0.19 6.6 70.0 31.5
0.26 F 11.1 F 51.3 F 27.6 F
F F F F
0.03 1.08 8.92 4.20
0.02a 2.20a 8.44a 3.24
33.6 F 1.11b,c 0.20 8.7 63.2 29.5
F F F F
0.02b 0.90c,d 5.46d 3.56
Values are means F SD for 8 rats. a P b 0.001 when compared with group C. b P b 0.001 when compared with group F. c P b 0.05 compared with group C. d P b 0.01 when compared with group F. Fig. 1. Liver histology in control rats (H & E; scale bar = 25 Am).
remarkable hypertriglyceridemia in both liver and plasma in our experimental model. Fructose-induced steatosis was also confirmed histologically (Fig. 2). In the F group, the levels of triglyceride in the plasma and liver were significantly increased compared to the control group (P b 0.001). Increase in plasma and liver triglyceride levels was observed to be less in the Ta1 injected group compared to the control group (P b 0.01). In the F group, the levels of TBARS in the liver were significantly increased compared to control rats (P b 0.001). In the F + T group, TBARS levels were significantly lower compared to the F group (P b 0.001) in the liver. In the F group, the level of NO in the liver was significantly increased compared to control rats (P b 0.001). In the F + T group, NO levels were significantly lower compared to the F group (P b 0.001). When the Ta1injected group was compared with the control group, NO and SOD values did not differ significantly, but TBARS and XO levels showed significant differences (P b 0.05). There were significant changes in most of the liver tissue enzymes, except GSH-Px. The oxidant enzyme XO activity increased significantly (P b 0.001) in the F group, whereas it was significantly lower in the F + T group compared to the F group (P b 0.001). The activity of SOD was significantly increased (P b 0.001) in the F group compared to controls and significantly lower (P b 0.01) in the F + T group compared to the F group (Table 1).
As shown in Table 2, the plasma IL-1h and IL-6 levels were significantly higher (P b 0.01 and P b 0.001, respectively) in the F group compared to control rats, and the levels of these cytokines were significantly lower (P b 0.001) in the F + T group compared to the F group. Although IL-1h and IL-6 levels decreased by injection of Ta1, they did not decrease to control levels (P b 0.01). The plasma IL-2 levels were also higher (P b 0.001) in the F group compared to control rats. IL-2 levels in the F + T group were higher than the other two groups. Histopathological findings The histology of the liver was normal in the control group (Fig. 1). In the F group, the most consistent findings in the histologic sections of liver tissues stained with hematoxylin and eosin were the macrovesicular and microvesicular steatosis in the liver tissues (Fig. 2). Ta1 treatment protected the majority of the liver cells. Nevertheless, minimal macrovesicular and microvesicular steatosis was observed in the remaining cells. In addition, a few cells were
Table 2 The plasma IL 1h, IL-2, and IL-6 levels in control, fructose-induced, and fructose-induced plus thymosin a1-treated groups Parameters Trigliserid (mg/dL) Trigliserid (mg/dL wet tissue) IL 1h (pg/mL) IL 2 (pg/mL) IL 6 (pg/mL)
C
F
F+T a
44.1 F 5.19 346.4 F 36.8
68.0 F 12.60 808.2 F 110.8a
21.4 F 5.82 15.7 F 3.49 9.4 F 1.15
41.5 F 3.97a 25.9 F 2.62a 19.6 F 2.01a
Values are means F SD for 8 rats. a P b 0.001 when compared with group C. b P b 0.001 when compared with group F. c P b 0.01 when compared with group C. d P b 0.01 when compared with group F.
46.8 F 5.46b 542.4 F 46.4b,c 33.9 F 4.11c,d 34.6 F 6.24b,c 14.5 F 2.70b,c
Fig. 2. After 10 days of fructose treatment, hepatocellular degeneration, macrovesicular (arrow) and microvesicular steatosis were observed in the hepatic lobule, in absence of any inflammation (H & E; scale bar = 25 Am).
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Fig. 3. Liver histology of thymosin-treated rats. Minimal degeneration of hepatocytes and microvesicular steatosis are seen. However, some macrovesicular steatosis (arrow) are still observed (H & E; scale bar = 25 Am).
indicated to have picnotic nucleus (Fig. 3). As shown in Table 3, Ta1 treatment protected the intensity of steatosis in the liver significantly in comparison with untreated highfructose-fed animals (P b 0.05). The area of macrovesicular steatosis was measured in the livers of the F and F + T groups. The percentage of the macrovesicular steatosis was calculated according to the results shown in Table 3. Fructose induction resulted in a significant increase of the area of macrovesicular steatosis. Ta1 treatment prevented macrovesicular steatosis in liver.
Discussion Nonalcoholic steatohepatitis (NASH) is one of the most common liver diseases encountered in the United States and Europe. Although this term refers to a spectrum of hepatic pathology that resembles alcoholic liver disease, it refers to a wide spectrum of liver damage, ranging from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis [2]. A dtwo-hitT concept of disease pathogenesis has been proposed. The first hit is steatosis, and this is postulated to sensitize the liver to the second hit, which may be oxidative stress or abnormal cytokine production. Oxidative stress and lipid peroxidation are candidates for the second hit in the pathogenesis of NASH. Both animal data and human studies have shown a link between NASH and oxidative stress and lipid peroxidation [26,27]. The underlying mechanisms for the detrimental consequences of a high-fructose diet in animal models are not clear. It is known that hepatic fatty acid synthase is induced and de novo fatty acid synthesis is increased in Wistar rats that have been fed fructose in drinking water [7,8]. It is now established that oxidative damage is present in several animal models of steatohepatitis. Oliveira et al. have found that oxidative stress increased in the experimental hepatic steatosis caused by a choline-deficient diet [5]. Fructose-rich diets have been shown to have deleterious metabolic effects, including
glucose intolerance, insulin resistance, dyslipidemia, and liver dysfunction. Some of these metabolic effects of fructose are attributed to its rapid hepatic uptake and the fact that it bypasses the phosphofructokinase regulatory step in glycolysis [28,29]. Fructose-induced increase in triacylglycerols may be due to stimulation of triacylglycerol synthesis. Moreover, the possibility exists that fructose feeding facilitates oxidative damage. Following fructose consumption, excesses of NADH and NADPH are known to occur during the metabolism of fructose via the pentose shunt and sorbitol pathway [30,31]. In addition, fructose feeding results in increased xanthine oxidase activity, and to glyceraldehyde production, which can induce free radicals. In the metabolism of hypoxanthine by the enzyme xanthine oxidase, both the superoxide radical and hydrogen peroxide can be generated [32,33]. Indeed, this study shows that fructose-fed rats were more susceptible to peroxidative damage as measured by thiobarbituric acid reactive species. Fructose feeding results in increased liver xanthine oxidase activity and nitric oxide production, which can induce ROS. NO may potentiate cytotoxicity by reaction with superoxide anion to form peroxynitrite, which then causes protein nitration and tissue injury. The finding that intrahepatic accumulation of nitrotyrosine is associated with the histological severity of NASH strongly suggests that oxidative injury may play a significant role in the pathogenesis of NASH [34,35]. However, in the presence of massive injury, greatly increased NO production might induce the hepatocytes to progress to irreversible channel necrosis and cell death [36]. On the other hand, Zhu and Fung [37] reported that NO protects against liver injury by scavenging lipid radicals and inhibiting the lipid peroxidation chain reaction. Inducible NO may initiate and play a key role in the metabolic and functional stress responses of hepatocytes against endotoxin and cytokines. Sass et al. [38] found that iNOS-derived NO regulates proinflammatory genes in vivo, contributing to inflammatory liver injury. Steatosis-induced lipid peroxidation and ROS can consume antioxidant enzymes and vitamins [12]. Depletion of these protective substances may hamper ROS inactivation and increase lipid peroxidation and ROS-mediated damage. Our results also show that, in fructose-fed rats, the balance between prooxidants and antioxidants is disturbed by the increased activities of antioxidant enzymes such as SOD and GSH-Px.
Table 3 Comparison area of the macrovesicular steatosis in the liver A (control), B (high-fructose-fed), and C (Ta1 treatment) groups C Mean area of macrovesicular steatosis in livers
0%
F
F+T a
12.36 F 1.23%
4.34 F 0.28%b,c
The Kruskal–Wallis test was used for statistical analysis. Values are expressed as mean F SD, and n = 50 liver preparations for all groups. a P b 0.001 when compared with group C. b P b 0.001 when compared with group F. c P b 0.01 when compared with group C.
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This finding may indicate that the hepatic antioxidant enzymatic defense system is impaired in NASH. The balance between oxidative stress and antioxidant defense mechanisms may be impaired by depletion of enzymatic antioxidants and increased plasma levels of TBARS and NO in patients with NASH [39]. Several experimental therapies, including treatment with bile acids, antibiotics, nutritional supplements, and antioxidants, have had anecdotal success in selected patients, but improved understanding of the pathogenesis and natural history of NASH will be required to develop generally effective therapy for the disorder [2,40]. The release of ROS is thought to be responsible for the general liver damage that occurs, as evidenced by the ability to attenuate hepatotoxicity by antioxidant treatment or cytokine neutralization [41]. In primary steatohepatitis, this may be due to several vicious cycles involving steatosis, lipid peroxidation, mitochondrial damage, enchanced ROS formation, increased TNF-a, and depletion of antioxidants. TNF-a impairs mitochondrial respiration and also causes opening of the mitochondrial permeability transition pore, which may further increase mitochondrial ROS formation and lipid peroxidation [12,39]. The release of TNF-a activates specific redox sensitive kinases that can upregulate proinflammatory pathways and enhance insulin resistance [42]. Finally, ROS-associated lipid peroxidation and cytokines may be involved in the inflammatory cell infiltrate, thus extensive ROS formation in secondary NASH could play a role in necroinflammation [11]. Interleukins are regulatory peptides that can be produced by virtually every nucleated cell in the body, including liver cells [43]. Most types of cells in the liver, including Kupffer cells, hepatocytes, and stellate cells, either synthesize or respond to cytokines, whereas proinflammatory cytokines such as TNF-a, interleukin-6, and interleukin-1h are mainly involved in cholestasis and the synthesis of acute-phase proteins by activated Kupffer cells and hepatocytes [44]. Altered Kupffer cell production of various cytokines is also likely to deplete the liver of certain populations of lymphocytes, thereby changing vulnerability to the hepatotoxic actions of agents that activate both innate and T-cellmediated immune responses [45]. Abnormal cytokine production in NASH patients may also be due to abnormal macrophage function. TNF-a initiates various intracellular signals that increase mitochondrial permeability and the release of reactive oxygen species [11]. Mitochondrial ROS formation is further increased, which could cause more lipid peroxidation, cytokine induction, and fibrogenesis [12]. Faure et al. reported that a high-fructose diet in rats leads to insulin resistance and a defect in the free radical defense system, increasing oxidative stress. According to their study with vitamin E supplementation, there was improvement in insulin sensitivity in fructose-fed rats [9]. It is clear that the oxidant/antioxidant balance plays an important role in regulation of cytokines. Increased levels of proinflammatory cytokines are found in both the plasma and liver in alcoholic
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hepatitis. The plasma IL-6 concentration appears to correlate well with the clinical severity and course of alcoholic hepatitis. Inflammatory cytokines, including IL-1 and TNFa, are thought to associate with the pathophysiology of liver disease [11,46]. IL-1 and IL-6 are responsible for many cellular events that are present in acute inflammation. These cytokines increase acute phase protein synthesis in hepatocytes. IL-1 may induce the formation of oxygen free radicals, which mediate the neutrophil–endothelium interaction causing cell dysfunction and destruction [47,48]. Antioxidants such as vitamin E, N-acetylcysteine, betaine, and others may be beneficial in the treatment of NASH [49]. Abdelmalek et al. reported that betaine (a metabolite of choline and antioxidant) treatment provides histological improvement in NASH [50]. Ta1 is a synthetically produced thymic peptide that has been shown to enhance the response of T cells and stimulate the production of endogenous interleukins and interferons. Ta1 promotes angiogenesis and wound healing [14,18]. It is able to enhance natural killer cell activity and it has been successfully tested in patients with chronic hepatitis B. Moreover, it has been proposed that Ta1 may modulate the action of IL-2 [51,52]. Parallel to this information, IL-2 levels were also shown in our study to be increased remarkably in the Ta1-treated group. The thymic hormones were reported to be effective on lipid peroxidation. AdemoWlu et al. have reported that plasma and erythrocyte TBARS levels significantly decreased in the Ta1-injected rats [53]. Tada et al. showed that thymic hormones increased GSH levels in tacrolimus-induced nephrotoxicity [54]. Our data indicate that Ta1-treated rats developed significantly less liver steatosis and lipid peroxidation. Oxidative damage is also prominent in the liver of humans with NASH, as well as in the histologically similar disorder of alcoholic hepatitis [11,55]. Histopathological examinations also demonstrated that Ta1 also effectively stabilizes the severity of hepatic lesions in the liver of fructose-induced rats. Triglycerides are the main component of accumulated fatty droplets. In the present study, both liver and fasting serum triacylglycerol levels were significantly elevated by dietary treatment. However, serum ALT levels also were not significantly increased in this study. Stravitz and Sanyal reported that a low normal ALT value does not guarantee freedom from underlying steatohepatitis with advanced fibrosis [56]. In conclusion, these data provide experimental evidence for a promising antioxidant and protective effect of Ta1 in fructose-induced steatosis. Taken together, the data presented here indicate that: (a) ROS may be involved in the mechanism of nonalcoholic steatohepatitis; (b) the generation of ROS not only induces lipid peroxidation, but also modulates the production of cytokines (especially proinflammatory); (c) lipid peroxidation and cytokines may play a role in fructose-induced hepatocellular injury and thymosin a1 may have some beneficial effects on the treatment of steatosis by normalizing blood cytokine levels and by substantially protecting liver cells against free radical injury.
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Altogether, future investigations are necessary to elucidate the role of thymosin a1 in NASH prevention, and further studies at different doses of thymosin a1 are needed to determine the most appropriate dosage.
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