© 2008 The Authors Doi: 10.1111/j.1742-7843.2008.00324.x Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 476–481
Trolox Down-Regulates Transforming Growth Factor-β and Prevents Experimental Cirrhosis
Blackwell Publishing Ltd
Marina Galicia-Moreno1, Adriana Rodríguez-Rivera1, Karina Reyes-Gordillo1, José Segovia2, Mineko Shibayama3, Víctor Tsutsumi3, Paula Vergara2, Mario G. Moreno1, Eduardo Fernández-Martínez4, Víctor M. Pérez-Álvarez1 and Pablo Muriel1 1
External Section of Pharmacology, 2Department of Physiology Biophysics and Neurosciences, and 3Department of Experimental Pathology, D.F., and 4Research Center in Reproductive Biology, Autonomous University of Hidalgo, Hidalgo, Mexico (Received April 18, 2008; Accepted June 3, 2008) Abstract: Cirrhosis is a very common disease and its treatment is limited due to lack of effective drugs. Some studies indicate that this disease is associated with oxidative stress. Therefore, we decided to study the effect of trolox, an effective antioxidant, on experimental cirrhosis. Cirrhosis was induced by CCl4 administration (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks) to Wistar male rats. Trolox was administered daily (50 mg/kg, orally). Fibrosis was assessed histologically and by measuring liver hydroxyproline content. Glutathione, lipid peroxidation and glycogen were measured in liver; serum markers of liver damage were also quantified. Transforming growth factor- β (TGF-β) was determined by Western blot and quantified densitometrically. Alkaline phosphatase, γ-glutamyl transpeptidase and alanine aminotransferase increased in the group receiving CCl4; trolox completely or partially prevented these alterations. Glycogen was almost depleted by CCl4 but was partially preserved by trolox. Lipid peroxidation increased while glutathione decreased by CCl4 administration; trolox corrected both effects. Histology showed thick bands of collagen, necrosis and distortion of the hepatic parenchyma in the CCl4 group, such effects were prevented by trolox. Hydroxyproline content increased 5-fold by CCl4, while the group receiving both CCl4 and trolox showed no significant difference compared to the control group. CCl4 increased 3-fold TGF-β, while trolox completely prevented this increase. We found that trolox effectively prevented cirrhosis induced with CCl4 in the rat. Our results suggest that the beneficial effects of trolox may be associated to its antioxidant properties and to its ability to reduce the profibrogenic cytokine TGF- β expression.
Liver cirrhosis, one of the main causes of death in many countries, is mainly characterized by the excessive deposition of collagen or fibrosis. Very few drugs have shown beneficial properties to ameliorate this disease [1]. Oxidative stress has been shown to play an important role in the establishment of such an ailment [2,3]. Oxidative stress may be involved in fibrogenesis by increasing harmful cytokines, in particular the most profibrogenic cytokine transforming growth factorβ (TGF-β) [4]. Therefore, antioxidant therapy may be very useful in the treatment of hepatic fibrosis and cirrhosis. Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid) is a water soluble analogue of vitamin E or α-tocopherol. Trolox was reported to be an effective preservative in animal fats and vegetable oils [5] and an excellent antioxidant in vitro [6,7]. In sodium dodecyl sulfate micelles, trolox was shown to scavenge peroxy radicals eight times better than α-tocopherol [8]. Trolox reduces the liver damage caused by bromobenzene, iodobenzene and dimethylmaleate intoxication in the rat [9], protects rat hepatocytes against oxyradical damage and the ischaemic rat liver from reperfusion injury [10]. More recently, the beneficial effects of trolox were demonstrated on sepsis-induced hepatic drug metabolizing
Author for correspondence: Pablo Muriel, External Section of Pharmacology, Cinvestav-I.P.N., Apdo. Postal 14-740, Mexico 07000, D.F., Mexico (fax +52 55 5747 3394, e-mail
[email protected]).
dysfunction [11], and Diaz et al. in 2007 found that trolox enhances the antilymphoma effects of arsenic trioxide, while protecting against liver toxicity [12]. In the present work, we sought to investigate if trolox was capable of preventing CCl4-induced cirrhosis. We found that trolox at least partially prevented all the parameters studied herein; most importantly, it completely prevented fibrosis. Prevention of fibrogenesis was accompanied by a sharp downregulation of TGF-β expression and antioxidant effects. Materials and Methods Chemicals. Trolox, chloramine-T, methyl cellosolve, sodium thiosulphate, p-dimethylaminobenzaldehyde, anthrone, carboxymethylcellulose, thiobarbarbituric acid, γ-glutamyl-p-nitroanilide, L-γ-glutamyl-pnitroaniline, p-nitrophenyl phosphate and bovine serum albumin were purchased from Sigma Chemical Company (St. Louis, MO, USA). Carbon tetrachloride, sodium acetate, sodium hydroxide, glacial acetic acid, hydrochloric acid, sulfuric acid, ethanol, methanol, potassium hydroxide and formaldehyde were obtained from J.T. Baker (Xalostoc, Mexico). Methods. Male Wistar rats weighing initially 90 –100 g and fed with a Purina chow rat diet ad libitum were used. Cirrhosis was produced by intraperitoneal administration of CCl4 (0.4 g/kg body weight) dissolved in mineral oil three times per week for 8 weeks. Four groups were performed. Group 1 (n = 8) consisted in control animals receiving the vehicle only (oil). In group 2 (n = 15), CCl4 was administered for 8 weeks. Animals in group 3 (n = 15) received
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TROLOX ON LIVER FIBROSIS trolox (50 mg/kg suspended in 0.7% carboxy methyl-cellulose orally for 8 weeks, daily) plus CCl4. The rats in group 4 (n = 8) received trolox only. The dose of trolox used was obtained from a previous report in which 50 mg/kg/day effectively prevented oxidative stress induced by acute cholestasis in the rat [13]. All the animals were killed under light ether anaesthesia 72 hr after the last dose of CCl4 or mineral oil. Blood was collected by cardiac puncture and the liver was rapidly removed. All samples were kept on ice until analysis. All animals received human care according to the institution’s guidelines and the Mexican Official Norm (NOM-062-ZOO-1999) regarding technical specifications for production, care and use of laboratory animals. Serum enzyme activities. Serum was obtained for determination of liver damage by measuring the activities of alanine aminotransferase (ALT) [14], alkaline phosphatase (AP) [15] and γ -glutamyl transpeptidase (γ-GTP) [16]. Assessment of lipid peroxidation. The extent of lipid peroxidation was esteemed in liver homogenates by the measurement of malondialdehyde formation using the thiobarbituric acid method [17]. Protein was determined according to Bradford [18] using bovine serum albumin as standard. Reduced glutathione determination in liver. Liver pieces (250 mg) were homogenized on ice using a polytron homogenizer. The solution used for homogenization consisted of 3.75 ml of the phosphateethylenediaminetetraacetic acid buffer, pH = 8 and 1 ml of 25% H3PO4, which was used as a protein precipitant. The total homogenate was centrifuged at 4° at 100,000 ×g for the assay of reduced glutathione (GSH). Determination of GSH was performed according to Hissin and Hilf [19]. Glycogen determination. Small pieces of liver (0.5 g) were separated for glycogen determination using the anthrone reagent according to Seifter et al. [20] Histology. Samples of liver were taken from all the animals and fixed with 10% formaldehyde in phosphate-buffered saline for 24 hr. Those tissue pieces were washed with tap water, dehydrated in alcohol and embedded in paraffin. Sections of 6–7 μm were mounted in glass slides and covered with silane. Haematoxylin and eosin as well as Masson’s trichromic stains were performed in each slide. Collagen quantification. Collagen concentrations were determined by measuring hydroxyproline content in fresh liver samples after digestion with acid as we have previously described [21]. Western blot assays. The TriPure reagent (Roche Diagnostics, Mannheim, Germany) was used to isolate total protein from samples of liver tissue. Fresh tissue was homogenized in 1 ml of TriPure reagent, then 0.2 ml of chloroform was added to homogenates and the lower phase was treated with isopropanol to precipitate total
protein. Samples were centrifuged at 10,500 ×g for 10 min. at 4°, and then three washes were performed with 0.3 M guanidine hydrochloride in 95% ethanol. A final wash was performed with 100% ethanol, samples were centrifuged as previously described [22] and the pellet resuspended in 1% sodium dodecyl sulfate. Volumes equivalent to 50 μ g of proteins (determined by the bicinchoninic acid method) were transferred to a 12% polyacrylamide gel; separated proteins were transferred onto Immun-Blot™ polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). Next, blots were blocked with 5% skim milk and 0.05% Tween-20 for 30 min. at room temperature and independently incubated overnight at 4° with antibodies selective against TGF-β (MAB 1032 from Chemicon International Inc., Temecula, CA, USA). The following day, the membranes were washed and then exposed to a secondary peroxidase-labelled antibody (Zymed, San Francisco, CA, USA) diluted 1:4000 in the blocking solution for 1 hr at room temperature. Blots were washed and protein developed using the Western lightning™ Chemiluminescence detection system (Perking Elmer LAS Inc., Boston, MA, USA). Blots were stripped and incubated with a monoclonal antibody directed against β-actin [23], which was used as a control to normalize cytokine protein expression levels. The procedure to strip membranes was as follows. Firstly, blots were washed four times with phosphate-saline buffer, pH 7.4 (0.015 M, 0.9% NaCl), then immersed in stripping buffer (2mercaptoethanol 100 mM, sodium dodecyl sulfate 2% and Tris-HCl 62.5 mM, pH 6.7) for 30 min. at 60° with gentle shaking, membranes were then washed five times with 0.05% Tween-20 in phosphate-saline buffer. Images were digitalized using the BioDoc-It System (UVP Inc., Upland, CA, USA) and then analysed densitometrically using the LabWorks 4.0 Image Acquisition and Analysis software (UVP Inc.) as previously described [22]. Statistical analysis. Data are expressed as mean values ± S.E.M. Comparisons were carried out by anova followed by Dunnett’s or Tukey’s test, as appropriate, using SigmaStat for Windows, version 2.0 (Jandel Corp., San Rafael, CA, USA). Differences were considered statistically significant when P < 0.05.
Results Serum markers of liver damage (AP, γ-GTP and ALT) increased significantly by the chronic administration of CCl4 (table 1). In the case of AP, the group receiving CCl4 plus trolox showed a lower value of this enzyme; however, the difference was not statistically significant when compared to either the control or CCl4 group. The increase in serum γ-GTP was completely prevented by trolox, while this compound preserved only partially the normal activity of ALT. The group receiving trolox alone showed normal values of these serum markers of liver damage.
Table 1. Enzyme activities determined in serum in the control group (control), CCl 4-treated rats (CCl4), CCl4-rats treated with trolox (CCl4 + trolox) and rats administered with trolox alone (Trolox). Group Parameter AP γ-GTP ALT
Control
CCl4
CCl4 + trolox
Trolox
65.27 ± 7.39 20.25 ± 2.18 21.63 ± 2.48
119.76 ± 16.27 33.30 ± 5.59a 64.16 ± 4.23a
93.86 ± 7.27 12.20 ± 0.82b 45.44 ± 6.36ab
57.77 ± 5.88 22.14 ± 1.39 23.56 ± 1.38
AP, alkaline phosphatase; γ-GTP, γ-glutamyl transpeptidase; ALT, alanino aminotransferase. ‘a’ means significantly different from control at P < 0.05; ‘b’ means significantly different from the CCl4-treated rats, P < 0.05. © 2008 The Authors Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 476–481
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Fig. 1. Liver glycogen content determined in the control rats (Control), CCl4-treated rats (CCl4), CCl4 plus trolox-treated rats (CCl4 + trolox) and rats receiving trolox alone (Trolox). Each bar represents the mean value of experiments performed in duplicate assays ± S.E.M. (n = 6). ‘a’ means significantly different from control, P < 0.05. ‘b’ means significantly different from the CCl4 group, P < 0.05.
Fig. 3. Reduced glutathione (GSH) determined in liver homogenates from the control rats (Control), CCl4-treated rats (CCl4), CCl4 plus trolox-treated rats (CCl4 + trolox) and rats receiving trolox alone (Trolox). Each bar represents the mean value of experiments performed in duplicate assays ± S.E.M. (n = 6). ‘a’ means significantly different from control, P < 0.05. ‘b’ means significantly different from the CCl4 group, P < 0.05.
The liver stores energy in the form of glycogen. Therefore, quantification of glycogen is an indicator of the ability of the organ to store energy. Figure 1 depicts that chronic CCl4 almost depletes glycogen. Interestingly, trolox prevents partially but significantly the drop in this parameter. Trolox by itself did not modify liver glycogen content. Liver damage is frequently associated with oxidative stress and reactive oxygen species [2,3]. When reactive oxygen species attack lipids, a process called lipid peroxidation occurs destroying membranes leading to cell death. One final product of this process is malondialdehyde; the measurement of malondialdehyde is a reliable marker of lipid peroxidation. As expected, malondialdehyde increased significantly in the group treated with CCl4; however, this increase was lower than expected, probably because animals were killed 72 hr after the last dose of CCl4 and during this time some malondialdehyde may be eliminated. It is worth noting that trolox completely prevented the increase in lipid peroxidation levels (fig. 2). Another indicator of oxidative stress is the concentration of GSH in the citosol of liver cells. As GSH is a hydrophilic
tripeptide, it is an indicator of the redox state of the citosol of the cells. Figure 3 shows a significant decrement of GSH caused by CCl4; again trolox completely prevented this effect. The group receiving trolox alone produced no modifications on the GSH content. Histologically, haematoxylin and eosin stains of liver samples (fig. 4) show that CCl4 produced a great disruption of the hepatic parenchyma and blood and bile duct structures; in addition, CCl4 intoxication was associated with infiltration of inflammatory cells and some degree of necrosis. Importantly, trolox preserved the normal architecture of the parenchyma. The most important feature of cirrhosis is fibrosis. Extracellular matrix accumulation was estimated by measuring hydroxyproline content in the liver (fig. 5) and by a specific stain, trichromic of Masson (fig. 6) in which bands of collagen fibres are seen in blue. Hydroxyproline increased around 6fold by CCl4 chronic treatment. This effect was completely prevented by trolox (despite that the hydroxyproline content looks higher in the CCl4 + trolox group than in the control group, there was no statistical difference between both groups). Histological analysis corroborates the biochemical findings: thick bands of collagen were observed in liver samples from rats treated with CCl4 (fig. 6B). In contrast, very few and thin collagen bands were seen in samples from the trolox + CCl4 group (fig. 6C). Trolox did not alter the hepatic parenchyma (fig. 6D) or the hydroxyproline content (fig. 5) when administered alone. The most profibrogenic factor is the cytokine TGF-β; therefore, we sought to investigate if the antifibrotic effects of trolox were associated with an effect on this mediator. Figure 7 shows a Western blot of TGF- β. CCl 4 induced a 3-fold increase in TGF-β protein. Interestingly, trolox completely prevented this increase and when administered alone decreased the normal levels of the cytokine.
Fig. 2. Liver lipid peroxidation determined as malondialdehyde (MDA) content in the control rats (Control), CCl4-treated rats (CCl4), CCl4 plus trolox-treated rats (CCl4 + trolox) and rats receiving trolox alone (Trolox). Each bar represents the mean value of experiments performed in duplicate assays ± S.E.M. (n = 6). ‘a’ means significantly different from control, P < 0.05.
Discussion Trolox treatment to CCl4 rats prevented the increases in serum markers of liver damage at various extents and
© 2008 The Authors Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 476–481
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Fig. 4. Haematoxylin and eosin staining of liver sections from: (A) control rats (vehicle); (B) CCl 4-treated rats; (C) CCl4-treated with trolox and (D) rats receiving trolox alone.
preserved partially the liver glycogen content. Lipid peroxidation and GSH content altered by CCl4 towards oxidative stress were normalized by trolox. The integrity of the hepatic parenchyma was highly distorted by CCl4, while trolox preserved the normal structure of the hepatic tissue. Collagen, measured by the liver hydroxyproline content and seen by specific stain was importantly increased by chronic CCl4 administration, but trolox prevented this effect. The profibrogenic cytokine TGF-β augmented nearly 4-fold by CCl4; trolox abolished this effect. Our results suggest that the beneficial effects of trolox may be associated to its antioxidant properties and to its ability to reduce hepatic TGF-β. Although extrapolation to human disease can not be performed
Fig. 5. Collagen content determined in livers from the control rats (Control), CCl4-treated rats (CCl4), CCl4 plus trolox-treated rats (CCl4 + trolox) and rats receiving trolox alone (Trolox). Each bar represents the mean value of experiments performed in duplicate assays ± S.E.M. (n = 6). ‘a’ means significantly different from control, P < 0.05. ‘b’ means significantly different from the CCl4 group, P < 0.05.
directly, the present results indicate that trolox may be studied in the clinical situation. Lamson and Wing [24] were the first to report that CCl4 intoxication produced cirrhosis; later, Cameron and Karunaratne [25] published a systematic study that established the morphology and showed the standard experimental conditions of the model. Nowadays, CCl4-induced cirrhosis is probably the most widely used model to reproduce cirrhosis in rats and mice [3]. Furthermore, Pérez-Tamayo [26] has shown that the CCl 4-induced cirrhosis shares several similarities with human alcoholic liver cirrhosis, making this model a suitable tool to investigate drugs with therapeutic potential on this disease. Trolox is the hydrophilic derivative of vitamin E and it has previously been reported that vitamin E protects against CCl4-induced chronic liver damage and cirrhosis [27]. Parola et al. [27] also reported that vitamin E did not interfere with metabolic activation of CCl4, indicating that oxidative process is essential in the development of the disease. In the present work, we show that trolox, besides its antioxidant properties, prevents TGF-β1 expression; this effect may also account for the antifibrotic properties of the compound. Mak et al. [28] showed that vitamin E did not protect against lipid peroxidation and hepatocellular damage in mice intoxicated with CCl4; several dissimilarities in the studies may explain differences in the results; they used vitamin E, mice and CCl4 was administered acutely at a high dose, while we utilized trolox, rats and CCl4 was administered chronically at a lower dose. In other works, trolox almost completely prevented both liver necrosis and lipid peroxidation induced by iodobenzene and bromobenzene
© 2008 The Authors Journal compilation © 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 476–481
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Fig. 6. Trichromic of Masson staining of liver sections from: (A) control rats (vehicle); (B) CCl 4-treated rats; (C) CCl4-treated with trolox and (D) rats receiving trolox alone.
[29,30]. In addition, trolox protects the liver from arsenic trioxide intoxication [12], and it has also shown beneficial effects on sepsis-induced hepatic drug metabolizing dysfunction [31] and protects against methanol-induced hepatic damage [32]. Chronic liver damage may lead to over-production of the extracellular matrix, resulting in a pathological state known as fibrosis. Hepatic stellate cells are known to play an important role in this process. The activation of hepatic stellate cells is associated with liver injury. Hepatic stellate
Fig. 7. Trolox blockade of transforming growth factor-β (TGF-β) protein in samples of liver tissue determined by Western blot analysis from control rats (Control), CCl4-treated rats (CCl4), CCl4 plus trolox-treated rats (CCl4 + trolox) and rats receiving trolox alone (Trolox). β-Actin was used as an internal control. Signal intensities were determined by densitometric analysis of treated blots and values calculated as the ratio of TGF-β/β-actin. Each bar represents the mean value ± S.E.M. of six rats; lower panel shows a representative blot. ‘a’ means significantly different from control, P < 0.05. ‘b’ means significantly different from the CCl4 group, P < 0.05.
cells change to a myofibroblast-like phenotype and become engaged in tissue repair, acting as one of the major components of liver fibrosis, by producing large amounts of extracellular matrix [33,34]. Furthermore, the sensitivity of these cells to inflammatory molecules and their capacity to secrete numerous cytokines also allows them to participate and control different aspects of liver inflammatory events [35]. Previous studies indicate the importance of oxidative stress, especially lipid peroxidation products, in the activation of hepatic stellate cells [36,37]. These substances have been associated with the excessive production of collagen by hepatic stellate cells, and it is known that lipid peroxidation products, such as malondialdehyde, activate the α2 (I) collagen promoter [38], showing a possible link between oxidative stress and liver fibrosis. Therefore, the antioxidant trolox, that effectively preserved malondialdehyde and GSH normal levels, could prevent hepatic stellate cell activation and cytokines (like TGF-β) and extracellular matrix production by these cells, and thus preventing the development of cirrhosis. Acknowledgements The authors express their gratitude to Mr. Benjamín Salinas Hernández, Mr. Ramón Hernández, Q. F. B. Silvia Galindo and M. V. Z. Ricardo Gaxiola, for their excellent technical assistance. Marina Galicia-Moreno was a fellow of Conacyt (204466). This work was supported in part by Conacyt 54756 (JS) and PROMEP/103.5/05/1919 grants (EF). References 1 Muriel P, Rivera-Espinoza Y. Beneficial drugs for liver diseases. J Appl Toxicol 2008;28:93 –103.
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