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Garlic extract prevents CCl4-induced liver fibrosis in rats: The role of tissue transglutaminase
Digestive and Liver Disease 42 (2010) 571–577
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Garlic extract prevents CCl4 -induced liver fibrosis in rats: The role of tissue transglutaminase Giuseppe D’Argenio a,∗ , Daniela Caterina Amoruso a , Giovanna Mazzone a , Paola Vitaglione b , Antonietta Romano a , Maria Teresa Ribecco a , Maria Rosaria D’Armiento c , Ernesto Mezza c , Filomena Morisco b , Vincenzo Fogliano b , Nicola Caporaso a a
Gastroenterology Unit, Department of Clinical and Experimental Medicine, Federico II University, Naples, Italy Department of Food Science, Federico II University, Naples, Italy c Department of Biomorphological Science, Federico II University, Naples, Italy b
a r t i c l e
i n f o
Article history: Received 24 July 2009 Accepted 4 November 2009 Available online 8 December 2009 Keywords: Cystamine Garlic extract Liver fibrosis Tissue transglutaminase
a b s t r a c t Background and aim: Tissue transglutaminase contributes to liver damage in the development of hepatic fibrosis. In a model of neurodegeneration, the therapeutic benefit of cystamine has been partly attributed to its inhibition of transglutaminase activity. Garlic extract contains many compounds structurally related to cystamine. We investigated the anti-fibrotic effect of garlic extract and cystamine as specific tissue transglutaminase inhibitors. Methods: Rat liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4 ) for 7 weeks. Cystamine or garlic extract was administrated by daily intraperitoneal injection, starting from the day after the first administration of CCl4 . Hepatic function, histology, tissue transglutaminase immunostaining and image analysis to quantify Red Sirius stained collagen deposition were examined. Reverse transcription-polymerase chain reaction to detect alpha-SMA, IL-1 and tissue transglutaminase expression and Western blot for tissue transglutaminase protein amount were performed. Transglutaminase activity was assayed on liver homogenates by a radio-enzymatic method. Results: Transglutaminase activity was increased in CCl4 group and reduced by cystamine and garlic extract (p < 0.05). Treatment with cystamine and garlic extract reduced the liver fibrosis and collagen deposition, particularly in the garlic extract group (p < 0.01). Moreover, the liver damage improved and serum alanine aminotransferase was decreased (p < 0.05). Tissue transglutaminase immunolocalised with collagen fibres and is mainly found in the ECM of damaged liver. Alpha-SMA, IL-1, tissue transglutaminase mRNA and tissue transglutaminase protein were down-regulated in the cystamine and garlic extract groups compared to controls. Conclusion: These findings concurrently suggest that transglutaminase may play a pivotal role in the pathogenesis of liver fibrosis and may identify garlic cystamine-like molecules as a potential therapeutic strategy in the treatment of liver injury. © 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Liver fibrosis is a wound-healing response to a variety of chronic stimuli such as viral infection (hepatitis B and C), chronic ethanol consumption, metabolic and toxic diseases and non-alcoholic liver steatohepatitis [1]. Hepatic fibrosis leads to cirrhosis and various complications and is associated with high morbidity and mortality [2]. Thus, it is essential to develop therapeutic strategies to counteract liver fibrosis [3].
∗ Corresponding author at: Dipartimento di Medicina Clinica e Sperimentale, Gastroenterologia, Facoltà di Medicina, Università Federico II, Via Pansini, 5, 80131, Napoli, Italy. Tel.: +39 081 7462706; fax: +39 081 7462739. E-mail address: [email protected] (G. D’Argenio).
Liver fibrosis is due to an imbalance between synthesis and degradation of the extracellular matrix (ECM) proteins [4], whose excessive deposition alters organ structure and function [5]. A major biomarker for liver fibrosis is tissue transglutaminase (tTG) or type II transglutaminase [6], a calcium-dependent enzyme which catalyses the formation of (␥-glutamyl) lysine cross-links or deamination of proteins. These covalent cross-links might determine the oligomerisation of substrate protein(s), which acquire the peculiar features of resistance to breakage and chemical attack [7]. Various collagen types and other extracellular proteins such as fibronectin, laminin, nidogen and transforming growth factor- are glutamyl substrates of tTG [8]. A role for tTG has also been proposed in the pathological accumulation of insoluble protein aggregates that occurs in
1590-8658/$36.00 © 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.dld.2009.11.002
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neurodegenerative diseases sharing several pathogenetical features with liver damage, such as Alzheimer and Huntington [9–11]. Recent studies have shown the ability of cystamine (CYS) [-mercaptoethanolamine disulphide] to ameliorate clinical and histopathological damage in an animal model of Huntington and liver diseases [12,13] via inhibition of tTG. Cysteamine (the reduced form of CYS) acts by competitive inhibition of tTG, resulting in the formation of -(␥-l-glutamyl)–cysteamine bonds [14]. The formation of such bonds would compete with the generation of other transglutaminase (TG)-catalysed reactions (i.e. polyamination, protein cross-linking and deamination proteins), which may contribute to neurodegeneration [12]. Despite its good efficacy as a TG inhibitor, CYS determines many undesirable side effects such as nausea and trouble walking, which highly limits the use in humans [11,12,15]. Owing to its content of organosulphur compounds, structurally similar to CYS and known to have many biological properties [16], we selected garlic (Allium sativum) as a potential natural source of CYS analogues. Organosulphur compounds of alliacee are a group of phytochemicals characterised by antioxidant properties [17]. In this study, a garlic extract (GE) was prepared, chemically characterised and tested in vitro and in vivo for its ability to modulate tTG activity and liver fibrosis.
The MS/MS detection was performed acquiring data in positive ion mode. The quantification was carried out in MRM (Multiple Reaction Monitoring). Ions corresponding to protonated molecules [M+H]+ for organosulphur compounds were monitored. Molecular weights and fragments of all monitored compounds were: m/z 178 → 88, 120 for alliin, 162 m/z → 76 for S-allylcysteine and 163 m/z → 88 for allicin, respectively. The ions produced in MS/MS were obtained through fragmentation by a specific collision energy (13 V) of a selected ion precursor, applying a voltage of 4500 V. The quantification of compounds was obtained using calibration curves obtained with pure alliin, S-allylcysteine and allicin (Exrasynthèse, Genay, France). The detection limit was 10 ng/g for all the compounds.
2. Materials and methods
Male Wistar albino rats (200–250 g) were housed in a room at a mean constant temperature of 22 ◦ C with a 12-h light–dark cycle, and free access to standard pellet chow and water. The study was approved by the Federico II University School of Veterinary Medicine Animal Care.
2.1. Preparation of GE Peeled garlic (500 g) was freeze dried and reduced to a fine powder in an electric mixer. The powder was transferred in a 1 L glass bottle and added with an equal volume (500 mL) of deionised water:ethanol 50:50 (v/v). Headspace was filled with a N2 atmosphere to reduce oxidation reaction of the extract and the bottle let in agitation for 12 h at 4 ◦ C in the dark. The mixture was filtered on a paper filter and the solution collected in a 1 L glass balloon. The GE was then evaporated under vacuum at room temperature until its volume was reduced to one half. Successively the extract was freeze dried, reduced to a fine powder by a mixer and stored at 4 ◦ C until the use. This procedure gave an extraction yield of 3.4%. 2.2. Chemical characterisation of GE The proximal composition of GE (carbohydrates, dietary fibre, protein and water) was carried out by official analytical methods. The Folin Ciocalteau method [18] was used for the quantification of total polyphenols. 2.3. Analysis of GE organosulphur compounds Identification and quantitative determination of the organosulphur compounds present in GE was carried out by HPLC/MS/MS. To this purpose an API 2000 triple quadrupole mass spectrometer (Applied Biosystem Sciex), with a Turboionspray (TIS) interface, coupled with an HPLC binary micropumps (Perkin Elmer, USA, mod. Series 200) was used. All the analyses were performed using drying gas (N2 ) at 300 ◦ C. Chromatographic separations of organosulphur compounds were performed on a Sphereclone 5 C18 ODS [2] (250 × 4.6 mm) (Phenomenex, Torrance, CA, USA), column using the following mobile phases, water (solvent A) and MeOH (solvent B) and a flow of 0.5 mL/min. Initial conditions were 90% A–10% B, then the following gradient elution was applied: 0–15 min 50% A–50% B, 15–20 min 50% A–50% B, 20–30 min 90% A–10% B.
2.4. In vitro effect of GE on TG activity To assess the effect of GE on TG activity, GE was incubated in vitro for 30 min either at room temperature or 37 ◦ C with normal rat liver homogenate (10%, w/v) as source of TG activity. The optimal concentration of GE was calculated to obtain an inhibition of TG activity of about 80% similar to that obtained with 1 mg/mL CYS. 2.5. Animals
2.6. Experimental model of progressive fibrosis Liver fibrosis was induced by intraperitoneal injection, twice a week, of carbon tetrachloride (CCl4 ) 0.2 mL/100 g in refined olive oil (1:1) for 7 weeks according to a well-established model [19]. A cohort of animals was injected with CCl4 for 7 weeks, after which animals were killed in a box for anaesthesia under CO2 atmosphere and their livers were harvested. Livers were harvested at peak fibrosis (3 days after the final injection of CCl4 ). After harvesting, livers were divided with a minimum of two lobes fixed in formalin for histologic analysis and immunohistochemistry and the remaining liver was snap-frozen for homogenisation to assay TG activity and to extract total liver RNA and total liver protein. Serum was also collected from each rat to analyse biochemical parameters. 2.7. Animal treatment and experimental groups Liver fibrosis was induced for 7 weeks in a set of experiments. Three experimental groups were designed as follows: (1) control group receiving ip injection of vehicle alone (n = 8); (2) rats receiving CYS (150 mg/kg b.w.) daily ip (n = 8); and (3) rats receiving GE (200 mg/kg b.w.) daily ip (n = 8). A group of five normal rats was also included in the study. 2.8. Histologic analysis Ten percent formalin-fixed paraffin-embedded sections of liver tissue were divided into 4 m sections by using routine techniques and mounted onto slides with coverslips. Representative sections of each fixed liver were stained with H&E and Sirius red/Fast green, according to standard protocols. All histologic analyses then were undertaken by an experienced histopathologist (M.R.D.) in a blinded manner.
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2.9. Immunohistochemistry To detect the immunohistochemical localisation of tTG, sections from formalin-fixed, paraffin-embedded specimens were deparaffinized and rehydrated in decreasing concentrations of ethyl alcohol. All tissue sections were incubated with fresh 3% hydrogen peroxide for 20 min, and then washed with phosphatebuffered saline (PBS) (0.1 mol/L phosphate and 0.15 mol/L sodium chloride, pH 7.4). The sections were sequentially incubated in 1% normal blocking serum for 30 min, and then with mouse monoclonal anti-tTG antibody (CUB 7402) (NeoMarkers, CA, USA) primary antibody for 60 min at room temperature. Subsequent incubations included appropriate biotinylated secondary antibody for 60 min and avidin–biotin–peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA, USA) for 30 min followed by the substrate, 3,3 diaminobenzidine (10 g/mL in Tris–HCl buffer, pH 7.6 containing 0.03% H2 O2 ), for 4 min. Negative controls were obtained by omitting the primary antibody. The sections, counterstained with haematoxylin, were then mounted and observed under light microscopy by a blinded pathologist. 2.10. Determination of liver collagen content For the detection and quantification of collagen, sections were stained with picrosirius red solution. The extent of liver fibrosis was determined as the proportion of picrosirius-stained area in each section. For each rat, 64 fields of a constant raster of 31 mm2 were analysed at 100-fold final magnification. For semiautomated morphometry a Sony 3CCD (model DXC-950P) videomicroscope equipped with motor stage and the Quantimed 500MC (Leica, Germany) software were used. 2.11. Determination of serum biochemical parameters Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin levels were determined to assess liver function by using a Modular Autoanalyzer (Roche Diagnostics, GmbH, D-68298, Mannheim, Germany). 2.12. TG activity TG activity (tTG) in liver homogenised in RIPA lysis buffer as described below, was assayed according to a modified version of the method described by Lorand et al. [20]: 30 L of sample was added to 45 L of reaction mixture containing a final concentration of 0.25 mM 14 C-putrescine (Amersham, UK), 50 mM dithiothreitol, 10 mM CaCl2 and 4% (w/v) dimethylcasein in Tris–HCl buffer (50 mM), pH 9.0, with 0.1% Triton X-100 and incubated with shaking at 37 ◦ C for 20 min. Then, 20 L was spotted onto 3MM Whatman round paper filters (2 cm) and immediately plunged into 10% ice cold trichloroacetic acid for 15 min. Two consecutive 15-min washings were performed in 5% ice cold trichloroacetic acid followed by a brief washing in ethanol–acetone (50%, v/v) and then in acetone. The dried paper filters were counted in 6 mL of Aquasure scintillant (Dupont-NEN). A similar procedure was adopted for blanks, standards and controls. TG units were expressed as 1 mU = 1 nmol of putrescine in acceptor protein at 37 ◦ C, pH 9. 2.13. Western blotting Liver samples were homogenised in RIPA lysis buffer (0.1% sodium dodecyl sulphate (SDS), 0.5% deoxycholate, 1% Nonidet, 100 mM NaCl, 10 mM Tris–HCl (pH 7.4)) containing a protease inhibitor cocktail (Sigma, St Louis, Missouri, USA), 0.5 mM dithiothreitol and 0.5% phenylmethylsulphonyl fluoride. Liver
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homogenates from rats of each experimental group as well as normal controls were run on 10% SDS/polyacrylamide gel according to Laemmli [21] and transferred to nitrocellulose using Biorad transblot apparatus. Anti-tTG mouse monoclonal antibody (CUB 7402; NeoMarkers, California, USA) diluted 1:1000 was added and incubation continued overnight at 4 ◦ C. The appropriate avidin conjugated secondary antibody was added and immunoreactive protein was detected by development with the ABC Vectastain kit (Vector Laboratories, Burlingame, California, USA) according to the manufacturer’s instructions. 2.14. RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from rat liver samples using Trizol reagent (Gibco Reagent, USA) according to the manufacturer’s protocol. mRNA levels of the gene under analysis was measured by semiquantitative RT-PCR amplification. Rat mRNA sequence was selected to design primer pairs for RTPCR reactions using the Oligo 4.05 software (National Biosciences Inc., Plymouth, Minnesota, USA). Primer sequences were as follows: for tTG, upper primer 5 -TGGCAGAGAAAGAGGAGACAGG3 , lower primer 5 -CATACAGGGAATCAGAAAGTGG-3 ; and for GAPDH, upper primer 5 -CACAGTCAAGGCTGAGAATG-3 , lower primer 5 -GCATTGCTGACAATCTTGAG-3 . Amplifications were carried out under the following conditions: 94 ◦ C for 60 s, 57 ◦ C for 60 s and 72 ◦ C for 60 s. To highlight the effect of GE, we also evaluated the expression of alpha-SMA as profibrogenic gene and IL-1 as pro-inflammatory gene in rat livers. Primer sequences were as follows: for alpha-SMA, upper primer 5 -CGATAGAACACGGCATCATC-3 lower primer 5 CATCAGGCAGTTCGTAGCTC-3 . Amplification was carried out under the following conditions: 94 ◦ C for 60 s, 61 ◦ C for 60 s and 72 ◦ C for 60 s. For IL-1, upper primer 5 -ACTTGGGCTGTCCAGATGAGA3 , lower primer 5 -GCCTGCAGTGCAGCTGTCTA-3 , amplification conditions were: 94 ◦ C for 60 s, 62 ◦ C for 60 s and 72 ◦ C for 60 s. According to the expression level of the target genes, the number of cycles was 35. The correct number of cycles was determined previously to ensure that PCR was taking place in the linear range in order to guarantee a proportional relationship between input RNA and densitometric readout. Each RT-PCR experiment was repeated at least three times. Amplification products were electrophoresed on 2% agarose gel in 1× TAE. Semiquantitative analysis of mRNA levels was carried out using the software associated with the Fluor S imager (BioRad Company, Hercules, California, USA). The density values of RT-PCR products were normalised with respect to endogenous GAPDH product levels. 2.15. Statistics Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Values of p < 0.05 were regarded as significant. 3. Results 3.1. Chemical characterisation of GE The chemical composition of the GE was reported in Table 1. The analysis of total polyphenols showed a content of these bioactive compounds of 0.3 mg gallic acid equivalent/g GE. As shown in Fig. 1, the HPLC/MS/MS analysis of organosulphur compounds of GE, allowed the identification of three main compounds. The amount of organosulphur compounds in GE was: alliin 54.2 g/g GE, S-allylcysteine 8 g/g GE and allicin 0.4 g/g GE.
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Table 1 Chemical composition of garlic extract (g/100 g). Water Carbohydrates Total dietary fibre Proteins Lipids Ashes
19.8 44.7 0.8 17.9 0.8 3.9
3.2. In vitro effect of GE on TG activity To assess the effect of GE on TG activity, GE was incubated in vitro with normal rat liver homogenate (10%, w/v) as source of tTG activity. The optimal concentration of GE was calculated to obtain an inhibition of TG activity similar to that obtained with 1 mg/mL CYS. Results are shown in Fig. 2. The GE inhibited about 80% of the enzyme activity when incubated with liver homogenate for 30 min at room temperature at the concentration of 4.3 mg/mL. Higher concentration of GE (8.6 mg/mL) inhibited only the 61% of tTG activity. The experiment was also carried out at 37 ◦ C with similar results (not shown). 3.3. Histology and liver collagen content All harvested livers were subjected to histologic analysis after H&E and picrosirius red/fast green staining by M.R.D. and E.M. in a blinded manner. Livers harvested from rats administering CCl4 for 7 weeks showed a pattern of fibrosis with septa linking the centrolob-
Fig. 2. In vitro inhibition of transglutaminase (TG) activity: liver homogenate as source of TG activity was incubated at room temperature for 30 min with cystamine or garlic extract at the indicated concentration. The first column represents the TG activity without inhibitors. Results are the mean of three different experiments.
ular veins. Sometimes livers showed the histologic appearance of an advanced fibrosis (Fig. 3G). Red Sirius staining of livers from rats treated with CCl4 showed a pattern of fibrosis with established septa linking hepatic veins and further new matrix bridging these areas to the portal tracts (Fig. 3A). CYS treatment significantly improved liver fibrosis showing only perivascular fibrosis with initial septa (Fig. 3B). Livers of rats treated with GE showed evidence of a residual perivenular fibrosis: there were no bridging septa from the hepatic veins to the portal tracts. In addition, the calibre of the remaining septa was reduced in width compared with peak fibrosis
Fig. 1. HPLC/MS/MS analysis of GE. Panel A shows the Total Ion Chromatogram (TIC). Peaks in panels B–D were obtained extracting the specific transitions of alliin, Sallylcysteine and allicin as reported in Section 2.
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Fig. 3. Representative Red Sirius staining, immunohistochemistry using tTG antibody and H&E on rat liver sections: (A) Red Sirius staining 7 weeks after CCl4 shows a fibrosis pattern with collagen deposition and porto-central septa (original magnification ×60). (B) Treatment with cystamine induces a significant improvement of liver fibrosis with only perivascular fibrosis and initial septa (original magnification ×60). (C) After treatment with garlic extract only residual perivenular fibrosis without septa was present. (D–F) tTG immunostaining clearly overlaps the Red Sirius pattern suggesting a co-localisation with newly formed collagen fibres (original magnification ×100). (G) Livers harvested from rats administering CCl4 for 7 weeks showed a pattern of fibrosis with septa linking the centrolobular veins. Sometimes livers showed the histologic appearance of an advanced fibrosis (original magnification ×40).
(Fig. 3C). The morphometric analysis of the collagen fibres analysed by Quantimed are reported in Fig. 4A. 3.4. Immunohistochemistry
ment significantly reduced TG activity levels. In a similar manner GE treatment inhibited TG activity (Fig. 4B).
3.7. Western blotting
To assess the distribution of tTG in the fibrotic liver and in the liver of animals treated with CYS or GE, serial sections were immunostained for tTG antibody. As shown in Fig. 3D, tTG immunolocalised with collagen fibres indicating that tTG is mainly found in the ECM of damaged liver. According to the low collagen deposition following CYS or GE treatments, the ECM was invariably almost negative for tTG staining (Fig. 3E and F).
tTG protein levels assayed by Western blotting are shown in Fig. 6A. Data revealed the increase of tTG protein expression in CCl4 compared with normal rats. tTG protein levels in either CYS or GE treated rats returned towards normal levels. These results were reproduced for all animals involved in the study in independent experiments.
3.5. Biochemical parameters
3.8. RNA extraction and RT-PCR
Serum AST, ALT, ALP and total bilirubin levels were determined to assess liver function. Rats with CCl4 -induced fibrosis showed serum levels of ALT and ALP higher than those of normal rats. Treatment with either CYS or GE reported these values within the normal range (Fig. 5). No statistically significant difference in total bilirubin concentration was found in the different groups compared with fibrosis control (not shown).
mRNA levels of tTG by semiquantitative RT-PCR were determined to assess whether expression of this gene was modified in the fibrotic liver. We found that tTG mRNA was more abundant in CCl4 fibrotic rats, increasing from two to four times compared with normal rats. mRNA levels of tTG in CYS and GE were decreased compared to control (CCl4 ) group. Accordingly, The expression of pro-fibrogenic gene alpha-SMA was up-regulated in CCl4 rats accounting for the increased collagen deposition. Alpha-SMA mRNA was decreased by CYS and more markedly by GE treatments. IL-1 was increased in CCl4 rat livers and down-regulated by CYS and GE treatments. Representative RT-PCR amplification patterns are shown in Fig. 6B–D.
3.6. TG activity TG activity assayed on liver homogenates by a radio-enzymatic method was increased after CCl4 treatment, whereas CYS treat-
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Fig. 4. (A) Percentage of collagen fibres quantified by morphometric analysis using Quantimed. The collagen deposition was significantly reduced by cystamine (CYS) and garlic extract (GE) treatments. (B) Transglutaminase (TG) activity assayed on liver homogenates by a radio-enzymatic method.
4. Discussion
Fig. 5. Liver function tests (AST, ALT, ALP). The ALT level increased in rat with CCl4 was reduced by cystamine (CYS) and garlic extract (GE) treatments.
Fig. 6. Effect of cystamine and garlic extract on tTG protein (A) and gene expression of tTG (B), alpha-SMA (C) and IL-1 (D) in rat liver. Representative Immunoblot and RT-PCR from liver tissues were obtained from normal rats and CCl4 , CCl4 + cystamine, CCl4 + garlic extract-treated rats.
The development of fibrosis, and particularly cirrhosis, is associated with a significant morbidity and mortality. Thus, it is extremely essential to develop anti-fibrotic strategies that are applicable to liver fibrosis. Most of the hypotheses relating to the pathogenesis of liver fibrosis have focused on the increased synthesis of the ECM or its decreased breakdown [4]. The resistance to the matrix degradation from specific collagenases may be due to a qualitative change of the matrix itself. This is possibly mediated by the tTG covalent stabilisation of the fibrils, incorporating ␥-(glutamyl) lysine cross-links. Eventually, this stabilisation moves the deposition–degradation balance towards accumulation and fibrosis [22]. The therapeutic benefit of CYS has been shown in several neurodegenerative disease models [9,11,23] and its efficacy has been attributed to the inhibition of tTG [12,14] and caspase-3 [24], as well as to the stimulation of glutathione (GSH) synthesis [25]. Cysteamine, a sulphide compound derived by CYS, has been recently demonstrated to be both an alternate amine-bearing substrate for tTG and an apparent competitive inhibitor of this enzyme [14]. Unfortunately, even at therapeutic doses, cysteamine gives undesirable effects that limits its use [12,15]; therefore, finding substitute compounds that may elicit cysteamine properties, but limiting side effects, may be an important challenge for clinicians. Garlic organosulphur compounds share many chemical properties with cysteamine and its disulphide CYS. In in vitro preliminary experiments, we observed that GE containing alliin, S-allylcysteine and allicin inhibits tTG activity in liver homogenate at the same extent as pure CYS despite the low concentration of 0.006% of GE (w/w). This suggests that the combination of these molecules may act in a synergistic manner compared to a single compound. Previously, the anti-fibrotic effects of garlic [26], GEs [17] and some garlic constituents [27] in different models of liver damage have been demonstrated by their antioxidant activity. Indeed, this is the first study to show that the prevention of liver injury by GE may be correlated to the direct inhibition of TG activity that in turn, reduces the levels of enzyme gene expression and transduction, possibly through a mechanism of negative feedback. It is known that in liver tissue TGF- stimulates the hepatic stellate cell differentiation into myofibroblasts that release collagen into the ECM where it is cross-linked by tTG. Furthermore, tTG activates latent TGF-, leading to de novo synthesis of tTG in a self-maintaining system. Data obtained from this study allows us
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to hypothesise that this mechanism, which leads to the formation of fibrosis, can be inhibited by garlic through the blockage of tTG activity and expression. The immunohistochemical study provided further insight into the role of tTG in fibrosis, showing that the enzyme localisation in damaged liver tissue acquires structural morphologies quite similar to that obtained by collagen deposition. Both tTG and collagen staining were strongly reduced by either CYS or GE treatment. This result was consistent with the finding of a down-regulation of alpha-SMA gene expression further supporting the antifibrogenic effect of GE, while the reduced IL-1 expression provides evidence for a role of GE also in reducing inflammation. Several recent studies reported health benefits of garlic diallylsulphides in many pathologies, such as cancer and CVD [28–30]. This study showed the ability of GE in reducing liver fibrosis and strengthening the therapeutic potential of the diallyldisulphide compound and allylmercaptan, which represents the main in vivo metabolite of organosulphur compounds [31]. Today, there is great public interest in natural remedies and in non-conventional therapies for the treatment of several diseases. Evidence supports the use of phytochemicals in combination with conventional therapies to reduce the side effects of pharmaceutical products, making garlic a potential candidate in this scenario. In spite of the numerous studies dealing with the beneficial effects of garlic consumption in humans, none had reported its ability in inhibiting tTG activity before our study. In conclusion, the data shown in this paper suggest that the inhibition of tTG activity obtained by a tailored GE preparation may allow recovery from liver injury and may block the progression of liver fibrosis. Since current therapeutic approaches in treating hepatic fibrosis are limited, a comprehensive understanding of the molecular mechanisms involved in tTG function and its inhibition would provide new therapeutic targets to inhibit the progression of this severe disease. Conflict of interest statement None declared. Acknowledgement D.C. Amoruso was recipient of a SIGE award, year 2008. This work was in part supported by a Campania Municipality grant on functional foods to VF. References [1] Török NJ. Recent advances in the pathogenesis and diagnosis of liver fibrosis. J Gastroenterol 2008;43:315–21. [2] Wanless IR, Nakashima E, Sherman M. Regression of human cirrhosis. Morphologic features and the genesis of incomplete septal cirrhosis. Arch Pathol Lab Med 2000;124:1599–607. [3] Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000;46:443–6. [4] Iredale JP, Benyon RC, Pickering J, et al. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest 1998;102:538–49.
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[5] Iredale JP. Hepatic stellate cell behaviour during resolution of liver injury. Semin Liver Dis 2001;21:427–36. [6] Elli L, Bergamini CM, Bardella MT, et al. Transglutaminases in inflammation and fibrosis of the gastrointestinal tract and the liver. Dig Liver Dis 2009;41:541–50. [7] Greenberg CS, Birckbichler PJ, Rice RH. Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. Fed Am Soc Exp Biol J 1992;5:3071–7. [8] Kojima S, Nara K, Rifkin DB. Requirement for transglutaminase in the activation of latent transforming growth factor-beta in bovine endothelial cells. J Cell Biol 1993;121:439–48. [9] Lorand L. Neurodegenerative diseases and transglutaminase. Proc Natl Acad Sci U S A 1997;93:14310–3. [10] Ikura K, Takahata K, Sasaki Y. Cross-linking of a synthetic partial length (1–28) peptide of the Alzheimer /A4 amyloid protein by transglutaminase. FEBS Lett 1993;326:109–11. [11] Lesort M, Tucholski J, Miller ML, et al. Tissue transglutaminase: a possible role in neurodegenerative diseases. Prog Neurobiol 2000;61:439–63. [12] Dedeoglu A, Kubilus JK, Jeitner TM, et al. Therapeutic effects of cystamine in a murine model of Huntington’s disease. J Neurosci 2002;22:8942–50. [13] Qiu J-F, Zhang Z-Q, Chen W, et al. Cystamine ameliorates liver fibrosis induced by carbon tetrachloride via inhibition of tissue transglutaminase. World J Gastroenterol 2007;28:4328–32. [14] Jeitner TM, Delikatny EJ, Ahlqvist J, et al. Mechanism for the inhibition of transglutaminase 2 by cystamine. Biochem Pharmacol 2005;69:961–70. [15] Dubinsky R, Gray C. CYTE-I-HD Phase I dose finding and tolerability study of cysteamine (Cystagon) in Huntington’s disease. Mov Disord 2006;21: 530–3. [16] Tapiero H, Townsend DM, Tew KD. Organosulfur compounds from alliaceae in the prevention of human pathologies. Biomed Pharmacother 2004;58:183–93. [17] Borek C. Antioxidant health effects of aged garlic extract. J Nutr 2001;131:1010S–5S. [18] Singleton VL, Rossi Jr JA. Colourimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am J Enol Viticult 1965;16: 144–58. [19] Issa R, Zhou X, Trim N, et al. Mutation in collagen-1 that confers resistance to the action of collagenase results in failure of recovery from CCl4 -induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. FASEB J 2003;17:47–9. [20] Lorand L, Campbell-Wilkes LK, Cooperstein L. A filter paper assay for transamidating enzyme using radioactive amine substrate. Ann Biochem 1972;50:623–31. [21] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. [22] Grenard P, Bresson-Hadni S, Alaoui SE, et al. Transglutaminase-mediated crosslinking is involved in the stabilization of extracellular matrix in human liver fibrosis. J Hepatol 2001;35:367–75. [23] Karpuj MV, BecherMW, Springer JE, et al. Prolonged survival and decreased abnormal movements in transgenic model of Huntington disease, with administration of the transglutaminase inhibitor cystamine. Nat Med 2002;8:143–9. [24] Lesort M, Lee M, Tucholski J, et al. Cystamine inhibits caspase activity. Implication for the treatment of polyglutamine disorders. J Biol Chem 2003;278:3825–30. [25] Chang ML, Klaidman LK, Adams Jr JD. The effects of oxidative stress on in vivo brain GSH turnover in young and mature mice. Mol Chem Neuropathol 1997;30:187–97. [26] Gedik N, Kabasakal L, S¸ehirli Ö, et al. Long-term administration of aqueous garlic extract (AGE) alleviates liver fibrosis and oxidative damage induced by biliary obstruction in rats. Life Sci 2005;76:2593–606. [27] Amagase H, Petesch B, Matsuura H, et al. Intake of garlic and its bioactive components. J Nutr 2001;131:955S–62S. [28] Powolny A, Singh SV. Multitargeted prevention and therapy of cancer by diallyl trisulfide and related Allium vegetable-derived organosulfur compounds. Cancer Lett 2008;269:305–14. [29] Kritchevsky D. The effect of dietary garlic on the development of cardiovascular disease. Trends Food Sci Technol 1991;2:141–4. [30] Ackerman R, Mulrow CD, Ramirez G, et al. Garlic shows promise for improving some cardiovascular risk factors. Arch Int Med 2001;161:813–24. [31] Egen-Schwind C, Eckard R, Kemper FH. Metabolism of garlic constituents in the isolated perfused rat liver. Planta Med 1992;58:301–5.
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Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld
Liver, Pancreas and Biliary Tract
Garlic extract prevents CCl4 -induced liver fibrosis in rats: The role of tissue transglutaminase Giuseppe D’Argenio a,∗ , Daniela Caterina Amoruso a , Giovanna Mazzone a , Paola Vitaglione b , Antonietta Romano a , Maria Teresa Ribecco a , Maria Rosaria D’Armiento c , Ernesto Mezza c , Filomena Morisco b , Vincenzo Fogliano b , Nicola Caporaso a a
Gastroenterology Unit, Department of Clinical and Experimental Medicine, Federico II University, Naples, Italy Department of Food Science, Federico II University, Naples, Italy c Department of Biomorphological Science, Federico II University, Naples, Italy b
a r t i c l e
i n f o
Article history: Received 24 July 2009 Accepted 4 November 2009 Available online 8 December 2009 Keywords: Cystamine Garlic extract Liver fibrosis Tissue transglutaminase
a b s t r a c t Background and aim: Tissue transglutaminase contributes to liver damage in the development of hepatic fibrosis. In a model of neurodegeneration, the therapeutic benefit of cystamine has been partly attributed to its inhibition of transglutaminase activity. Garlic extract contains many compounds structurally related to cystamine. We investigated the anti-fibrotic effect of garlic extract and cystamine as specific tissue transglutaminase inhibitors. Methods: Rat liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl4 ) for 7 weeks. Cystamine or garlic extract was administrated by daily intraperitoneal injection, starting from the day after the first administration of CCl4 . Hepatic function, histology, tissue transglutaminase immunostaining and image analysis to quantify Red Sirius stained collagen deposition were examined. Reverse transcription-polymerase chain reaction to detect alpha-SMA, IL-1 and tissue transglutaminase expression and Western blot for tissue transglutaminase protein amount were performed. Transglutaminase activity was assayed on liver homogenates by a radio-enzymatic method. Results: Transglutaminase activity was increased in CCl4 group and reduced by cystamine and garlic extract (p < 0.05). Treatment with cystamine and garlic extract reduced the liver fibrosis and collagen deposition, particularly in the garlic extract group (p < 0.01). Moreover, the liver damage improved and serum alanine aminotransferase was decreased (p < 0.05). Tissue transglutaminase immunolocalised with collagen fibres and is mainly found in the ECM of damaged liver. Alpha-SMA, IL-1, tissue transglutaminase mRNA and tissue transglutaminase protein were down-regulated in the cystamine and garlic extract groups compared to controls. Conclusion: These findings concurrently suggest that transglutaminase may play a pivotal role in the pathogenesis of liver fibrosis and may identify garlic cystamine-like molecules as a potential therapeutic strategy in the treatment of liver injury. © 2009 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
1. Introduction Liver fibrosis is a wound-healing response to a variety of chronic stimuli such as viral infection (hepatitis B and C), chronic ethanol consumption, metabolic and toxic diseases and non-alcoholic liver steatohepatitis [1]. Hepatic fibrosis leads to cirrhosis and various complications and is associated with high morbidity and mortality [2]. Thus, it is essential to develop therapeutic strategies to counteract liver fibrosis [3].
∗ Corresponding author at: Dipartimento di Medicina Clinica e Sperimentale, Gastroenterologia, Facoltà di Medicina, Università Federico II, Via Pansini, 5, 80131, Napoli, Italy. Tel.: +39 081 7462706; fax: +39 081 7462739. E-mail address: [email protected] (G. D’Argenio).
Liver fibrosis is due to an imbalance between synthesis and degradation of the extracellular matrix (ECM) proteins [4], whose excessive deposition alters organ structure and function [5]. A major biomarker for liver fibrosis is tissue transglutaminase (tTG) or type II transglutaminase [6], a calcium-dependent enzyme which catalyses the formation of (␥-glutamyl) lysine cross-links or deamination of proteins. These covalent cross-links might determine the oligomerisation of substrate protein(s), which acquire the peculiar features of resistance to breakage and chemical attack [7]. Various collagen types and other extracellular proteins such as fibronectin, laminin, nidogen and transforming growth factor- are glutamyl substrates of tTG [8]. A role for tTG has also been proposed in the pathological accumulation of insoluble protein aggregates that occurs in
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neurodegenerative diseases sharing several pathogenetical features with liver damage, such as Alzheimer and Huntington [9–11]. Recent studies have shown the ability of cystamine (CYS) [-mercaptoethanolamine disulphide] to ameliorate clinical and histopathological damage in an animal model of Huntington and liver diseases [12,13] via inhibition of tTG. Cysteamine (the reduced form of CYS) acts by competitive inhibition of tTG, resulting in the formation of -(␥-l-glutamyl)–cysteamine bonds [14]. The formation of such bonds would compete with the generation of other transglutaminase (TG)-catalysed reactions (i.e. polyamination, protein cross-linking and deamination proteins), which may contribute to neurodegeneration [12]. Despite its good efficacy as a TG inhibitor, CYS determines many undesirable side effects such as nausea and trouble walking, which highly limits the use in humans [11,12,15]. Owing to its content of organosulphur compounds, structurally similar to CYS and known to have many biological properties [16], we selected garlic (Allium sativum) as a potential natural source of CYS analogues. Organosulphur compounds of alliacee are a group of phytochemicals characterised by antioxidant properties [17]. In this study, a garlic extract (GE) was prepared, chemically characterised and tested in vitro and in vivo for its ability to modulate tTG activity and liver fibrosis.
The MS/MS detection was performed acquiring data in positive ion mode. The quantification was carried out in MRM (Multiple Reaction Monitoring). Ions corresponding to protonated molecules [M+H]+ for organosulphur compounds were monitored. Molecular weights and fragments of all monitored compounds were: m/z 178 → 88, 120 for alliin, 162 m/z → 76 for S-allylcysteine and 163 m/z → 88 for allicin, respectively. The ions produced in MS/MS were obtained through fragmentation by a specific collision energy (13 V) of a selected ion precursor, applying a voltage of 4500 V. The quantification of compounds was obtained using calibration curves obtained with pure alliin, S-allylcysteine and allicin (Exrasynthèse, Genay, France). The detection limit was 10 ng/g for all the compounds.
2. Materials and methods
Male Wistar albino rats (200–250 g) were housed in a room at a mean constant temperature of 22 ◦ C with a 12-h light–dark cycle, and free access to standard pellet chow and water. The study was approved by the Federico II University School of Veterinary Medicine Animal Care.
2.1. Preparation of GE Peeled garlic (500 g) was freeze dried and reduced to a fine powder in an electric mixer. The powder was transferred in a 1 L glass bottle and added with an equal volume (500 mL) of deionised water:ethanol 50:50 (v/v). Headspace was filled with a N2 atmosphere to reduce oxidation reaction of the extract and the bottle let in agitation for 12 h at 4 ◦ C in the dark. The mixture was filtered on a paper filter and the solution collected in a 1 L glass balloon. The GE was then evaporated under vacuum at room temperature until its volume was reduced to one half. Successively the extract was freeze dried, reduced to a fine powder by a mixer and stored at 4 ◦ C until the use. This procedure gave an extraction yield of 3.4%. 2.2. Chemical characterisation of GE The proximal composition of GE (carbohydrates, dietary fibre, protein and water) was carried out by official analytical methods. The Folin Ciocalteau method [18] was used for the quantification of total polyphenols. 2.3. Analysis of GE organosulphur compounds Identification and quantitative determination of the organosulphur compounds present in GE was carried out by HPLC/MS/MS. To this purpose an API 2000 triple quadrupole mass spectrometer (Applied Biosystem Sciex), with a Turboionspray (TIS) interface, coupled with an HPLC binary micropumps (Perkin Elmer, USA, mod. Series 200) was used. All the analyses were performed using drying gas (N2 ) at 300 ◦ C. Chromatographic separations of organosulphur compounds were performed on a Sphereclone 5 C18 ODS [2] (250 × 4.6 mm) (Phenomenex, Torrance, CA, USA), column using the following mobile phases, water (solvent A) and MeOH (solvent B) and a flow of 0.5 mL/min. Initial conditions were 90% A–10% B, then the following gradient elution was applied: 0–15 min 50% A–50% B, 15–20 min 50% A–50% B, 20–30 min 90% A–10% B.
2.4. In vitro effect of GE on TG activity To assess the effect of GE on TG activity, GE was incubated in vitro for 30 min either at room temperature or 37 ◦ C with normal rat liver homogenate (10%, w/v) as source of TG activity. The optimal concentration of GE was calculated to obtain an inhibition of TG activity of about 80% similar to that obtained with 1 mg/mL CYS. 2.5. Animals
2.6. Experimental model of progressive fibrosis Liver fibrosis was induced by intraperitoneal injection, twice a week, of carbon tetrachloride (CCl4 ) 0.2 mL/100 g in refined olive oil (1:1) for 7 weeks according to a well-established model [19]. A cohort of animals was injected with CCl4 for 7 weeks, after which animals were killed in a box for anaesthesia under CO2 atmosphere and their livers were harvested. Livers were harvested at peak fibrosis (3 days after the final injection of CCl4 ). After harvesting, livers were divided with a minimum of two lobes fixed in formalin for histologic analysis and immunohistochemistry and the remaining liver was snap-frozen for homogenisation to assay TG activity and to extract total liver RNA and total liver protein. Serum was also collected from each rat to analyse biochemical parameters. 2.7. Animal treatment and experimental groups Liver fibrosis was induced for 7 weeks in a set of experiments. Three experimental groups were designed as follows: (1) control group receiving ip injection of vehicle alone (n = 8); (2) rats receiving CYS (150 mg/kg b.w.) daily ip (n = 8); and (3) rats receiving GE (200 mg/kg b.w.) daily ip (n = 8). A group of five normal rats was also included in the study. 2.8. Histologic analysis Ten percent formalin-fixed paraffin-embedded sections of liver tissue were divided into 4 m sections by using routine techniques and mounted onto slides with coverslips. Representative sections of each fixed liver were stained with H&E and Sirius red/Fast green, according to standard protocols. All histologic analyses then were undertaken by an experienced histopathologist (M.R.D.) in a blinded manner.
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2.9. Immunohistochemistry To detect the immunohistochemical localisation of tTG, sections from formalin-fixed, paraffin-embedded specimens were deparaffinized and rehydrated in decreasing concentrations of ethyl alcohol. All tissue sections were incubated with fresh 3% hydrogen peroxide for 20 min, and then washed with phosphatebuffered saline (PBS) (0.1 mol/L phosphate and 0.15 mol/L sodium chloride, pH 7.4). The sections were sequentially incubated in 1% normal blocking serum for 30 min, and then with mouse monoclonal anti-tTG antibody (CUB 7402) (NeoMarkers, CA, USA) primary antibody for 60 min at room temperature. Subsequent incubations included appropriate biotinylated secondary antibody for 60 min and avidin–biotin–peroxidase complex (ABC kit; Vector Laboratories, Burlingame, CA, USA) for 30 min followed by the substrate, 3,3 diaminobenzidine (10 g/mL in Tris–HCl buffer, pH 7.6 containing 0.03% H2 O2 ), for 4 min. Negative controls were obtained by omitting the primary antibody. The sections, counterstained with haematoxylin, were then mounted and observed under light microscopy by a blinded pathologist. 2.10. Determination of liver collagen content For the detection and quantification of collagen, sections were stained with picrosirius red solution. The extent of liver fibrosis was determined as the proportion of picrosirius-stained area in each section. For each rat, 64 fields of a constant raster of 31 mm2 were analysed at 100-fold final magnification. For semiautomated morphometry a Sony 3CCD (model DXC-950P) videomicroscope equipped with motor stage and the Quantimed 500MC (Leica, Germany) software were used. 2.11. Determination of serum biochemical parameters Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and total bilirubin levels were determined to assess liver function by using a Modular Autoanalyzer (Roche Diagnostics, GmbH, D-68298, Mannheim, Germany). 2.12. TG activity TG activity (tTG) in liver homogenised in RIPA lysis buffer as described below, was assayed according to a modified version of the method described by Lorand et al. [20]: 30 L of sample was added to 45 L of reaction mixture containing a final concentration of 0.25 mM 14 C-putrescine (Amersham, UK), 50 mM dithiothreitol, 10 mM CaCl2 and 4% (w/v) dimethylcasein in Tris–HCl buffer (50 mM), pH 9.0, with 0.1% Triton X-100 and incubated with shaking at 37 ◦ C for 20 min. Then, 20 L was spotted onto 3MM Whatman round paper filters (2 cm) and immediately plunged into 10% ice cold trichloroacetic acid for 15 min. Two consecutive 15-min washings were performed in 5% ice cold trichloroacetic acid followed by a brief washing in ethanol–acetone (50%, v/v) and then in acetone. The dried paper filters were counted in 6 mL of Aquasure scintillant (Dupont-NEN). A similar procedure was adopted for blanks, standards and controls. TG units were expressed as 1 mU = 1 nmol of putrescine in acceptor protein at 37 ◦ C, pH 9. 2.13. Western blotting Liver samples were homogenised in RIPA lysis buffer (0.1% sodium dodecyl sulphate (SDS), 0.5% deoxycholate, 1% Nonidet, 100 mM NaCl, 10 mM Tris–HCl (pH 7.4)) containing a protease inhibitor cocktail (Sigma, St Louis, Missouri, USA), 0.5 mM dithiothreitol and 0.5% phenylmethylsulphonyl fluoride. Liver
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homogenates from rats of each experimental group as well as normal controls were run on 10% SDS/polyacrylamide gel according to Laemmli [21] and transferred to nitrocellulose using Biorad transblot apparatus. Anti-tTG mouse monoclonal antibody (CUB 7402; NeoMarkers, California, USA) diluted 1:1000 was added and incubation continued overnight at 4 ◦ C. The appropriate avidin conjugated secondary antibody was added and immunoreactive protein was detected by development with the ABC Vectastain kit (Vector Laboratories, Burlingame, California, USA) according to the manufacturer’s instructions. 2.14. RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from rat liver samples using Trizol reagent (Gibco Reagent, USA) according to the manufacturer’s protocol. mRNA levels of the gene under analysis was measured by semiquantitative RT-PCR amplification. Rat mRNA sequence was selected to design primer pairs for RTPCR reactions using the Oligo 4.05 software (National Biosciences Inc., Plymouth, Minnesota, USA). Primer sequences were as follows: for tTG, upper primer 5 -TGGCAGAGAAAGAGGAGACAGG3 , lower primer 5 -CATACAGGGAATCAGAAAGTGG-3 ; and for GAPDH, upper primer 5 -CACAGTCAAGGCTGAGAATG-3 , lower primer 5 -GCATTGCTGACAATCTTGAG-3 . Amplifications were carried out under the following conditions: 94 ◦ C for 60 s, 57 ◦ C for 60 s and 72 ◦ C for 60 s. To highlight the effect of GE, we also evaluated the expression of alpha-SMA as profibrogenic gene and IL-1 as pro-inflammatory gene in rat livers. Primer sequences were as follows: for alpha-SMA, upper primer 5 -CGATAGAACACGGCATCATC-3 lower primer 5 CATCAGGCAGTTCGTAGCTC-3 . Amplification was carried out under the following conditions: 94 ◦ C for 60 s, 61 ◦ C for 60 s and 72 ◦ C for 60 s. For IL-1, upper primer 5 -ACTTGGGCTGTCCAGATGAGA3 , lower primer 5 -GCCTGCAGTGCAGCTGTCTA-3 , amplification conditions were: 94 ◦ C for 60 s, 62 ◦ C for 60 s and 72 ◦ C for 60 s. According to the expression level of the target genes, the number of cycles was 35. The correct number of cycles was determined previously to ensure that PCR was taking place in the linear range in order to guarantee a proportional relationship between input RNA and densitometric readout. Each RT-PCR experiment was repeated at least three times. Amplification products were electrophoresed on 2% agarose gel in 1× TAE. Semiquantitative analysis of mRNA levels was carried out using the software associated with the Fluor S imager (BioRad Company, Hercules, California, USA). The density values of RT-PCR products were normalised with respect to endogenous GAPDH product levels. 2.15. Statistics Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Values of p < 0.05 were regarded as significant. 3. Results 3.1. Chemical characterisation of GE The chemical composition of the GE was reported in Table 1. The analysis of total polyphenols showed a content of these bioactive compounds of 0.3 mg gallic acid equivalent/g GE. As shown in Fig. 1, the HPLC/MS/MS analysis of organosulphur compounds of GE, allowed the identification of three main compounds. The amount of organosulphur compounds in GE was: alliin 54.2 g/g GE, S-allylcysteine 8 g/g GE and allicin 0.4 g/g GE.
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Table 1 Chemical composition of garlic extract (g/100 g). Water Carbohydrates Total dietary fibre Proteins Lipids Ashes
19.8 44.7 0.8 17.9 0.8 3.9
3.2. In vitro effect of GE on TG activity To assess the effect of GE on TG activity, GE was incubated in vitro with normal rat liver homogenate (10%, w/v) as source of tTG activity. The optimal concentration of GE was calculated to obtain an inhibition of TG activity similar to that obtained with 1 mg/mL CYS. Results are shown in Fig. 2. The GE inhibited about 80% of the enzyme activity when incubated with liver homogenate for 30 min at room temperature at the concentration of 4.3 mg/mL. Higher concentration of GE (8.6 mg/mL) inhibited only the 61% of tTG activity. The experiment was also carried out at 37 ◦ C with similar results (not shown). 3.3. Histology and liver collagen content All harvested livers were subjected to histologic analysis after H&E and picrosirius red/fast green staining by M.R.D. and E.M. in a blinded manner. Livers harvested from rats administering CCl4 for 7 weeks showed a pattern of fibrosis with septa linking the centrolob-
Fig. 2. In vitro inhibition of transglutaminase (TG) activity: liver homogenate as source of TG activity was incubated at room temperature for 30 min with cystamine or garlic extract at the indicated concentration. The first column represents the TG activity without inhibitors. Results are the mean of three different experiments.
ular veins. Sometimes livers showed the histologic appearance of an advanced fibrosis (Fig. 3G). Red Sirius staining of livers from rats treated with CCl4 showed a pattern of fibrosis with established septa linking hepatic veins and further new matrix bridging these areas to the portal tracts (Fig. 3A). CYS treatment significantly improved liver fibrosis showing only perivascular fibrosis with initial septa (Fig. 3B). Livers of rats treated with GE showed evidence of a residual perivenular fibrosis: there were no bridging septa from the hepatic veins to the portal tracts. In addition, the calibre of the remaining septa was reduced in width compared with peak fibrosis
Fig. 1. HPLC/MS/MS analysis of GE. Panel A shows the Total Ion Chromatogram (TIC). Peaks in panels B–D were obtained extracting the specific transitions of alliin, Sallylcysteine and allicin as reported in Section 2.
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Fig. 3. Representative Red Sirius staining, immunohistochemistry using tTG antibody and H&E on rat liver sections: (A) Red Sirius staining 7 weeks after CCl4 shows a fibrosis pattern with collagen deposition and porto-central septa (original magnification ×60). (B) Treatment with cystamine induces a significant improvement of liver fibrosis with only perivascular fibrosis and initial septa (original magnification ×60). (C) After treatment with garlic extract only residual perivenular fibrosis without septa was present. (D–F) tTG immunostaining clearly overlaps the Red Sirius pattern suggesting a co-localisation with newly formed collagen fibres (original magnification ×100). (G) Livers harvested from rats administering CCl4 for 7 weeks showed a pattern of fibrosis with septa linking the centrolobular veins. Sometimes livers showed the histologic appearance of an advanced fibrosis (original magnification ×40).
(Fig. 3C). The morphometric analysis of the collagen fibres analysed by Quantimed are reported in Fig. 4A. 3.4. Immunohistochemistry
ment significantly reduced TG activity levels. In a similar manner GE treatment inhibited TG activity (Fig. 4B).
3.7. Western blotting
To assess the distribution of tTG in the fibrotic liver and in the liver of animals treated with CYS or GE, serial sections were immunostained for tTG antibody. As shown in Fig. 3D, tTG immunolocalised with collagen fibres indicating that tTG is mainly found in the ECM of damaged liver. According to the low collagen deposition following CYS or GE treatments, the ECM was invariably almost negative for tTG staining (Fig. 3E and F).
tTG protein levels assayed by Western blotting are shown in Fig. 6A. Data revealed the increase of tTG protein expression in CCl4 compared with normal rats. tTG protein levels in either CYS or GE treated rats returned towards normal levels. These results were reproduced for all animals involved in the study in independent experiments.
3.5. Biochemical parameters
3.8. RNA extraction and RT-PCR
Serum AST, ALT, ALP and total bilirubin levels were determined to assess liver function. Rats with CCl4 -induced fibrosis showed serum levels of ALT and ALP higher than those of normal rats. Treatment with either CYS or GE reported these values within the normal range (Fig. 5). No statistically significant difference in total bilirubin concentration was found in the different groups compared with fibrosis control (not shown).
mRNA levels of tTG by semiquantitative RT-PCR were determined to assess whether expression of this gene was modified in the fibrotic liver. We found that tTG mRNA was more abundant in CCl4 fibrotic rats, increasing from two to four times compared with normal rats. mRNA levels of tTG in CYS and GE were decreased compared to control (CCl4 ) group. Accordingly, The expression of pro-fibrogenic gene alpha-SMA was up-regulated in CCl4 rats accounting for the increased collagen deposition. Alpha-SMA mRNA was decreased by CYS and more markedly by GE treatments. IL-1 was increased in CCl4 rat livers and down-regulated by CYS and GE treatments. Representative RT-PCR amplification patterns are shown in Fig. 6B–D.
3.6. TG activity TG activity assayed on liver homogenates by a radio-enzymatic method was increased after CCl4 treatment, whereas CYS treat-
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Fig. 4. (A) Percentage of collagen fibres quantified by morphometric analysis using Quantimed. The collagen deposition was significantly reduced by cystamine (CYS) and garlic extract (GE) treatments. (B) Transglutaminase (TG) activity assayed on liver homogenates by a radio-enzymatic method.
4. Discussion
Fig. 5. Liver function tests (AST, ALT, ALP). The ALT level increased in rat with CCl4 was reduced by cystamine (CYS) and garlic extract (GE) treatments.
Fig. 6. Effect of cystamine and garlic extract on tTG protein (A) and gene expression of tTG (B), alpha-SMA (C) and IL-1 (D) in rat liver. Representative Immunoblot and RT-PCR from liver tissues were obtained from normal rats and CCl4 , CCl4 + cystamine, CCl4 + garlic extract-treated rats.
The development of fibrosis, and particularly cirrhosis, is associated with a significant morbidity and mortality. Thus, it is extremely essential to develop anti-fibrotic strategies that are applicable to liver fibrosis. Most of the hypotheses relating to the pathogenesis of liver fibrosis have focused on the increased synthesis of the ECM or its decreased breakdown [4]. The resistance to the matrix degradation from specific collagenases may be due to a qualitative change of the matrix itself. This is possibly mediated by the tTG covalent stabilisation of the fibrils, incorporating ␥-(glutamyl) lysine cross-links. Eventually, this stabilisation moves the deposition–degradation balance towards accumulation and fibrosis [22]. The therapeutic benefit of CYS has been shown in several neurodegenerative disease models [9,11,23] and its efficacy has been attributed to the inhibition of tTG [12,14] and caspase-3 [24], as well as to the stimulation of glutathione (GSH) synthesis [25]. Cysteamine, a sulphide compound derived by CYS, has been recently demonstrated to be both an alternate amine-bearing substrate for tTG and an apparent competitive inhibitor of this enzyme [14]. Unfortunately, even at therapeutic doses, cysteamine gives undesirable effects that limits its use [12,15]; therefore, finding substitute compounds that may elicit cysteamine properties, but limiting side effects, may be an important challenge for clinicians. Garlic organosulphur compounds share many chemical properties with cysteamine and its disulphide CYS. In in vitro preliminary experiments, we observed that GE containing alliin, S-allylcysteine and allicin inhibits tTG activity in liver homogenate at the same extent as pure CYS despite the low concentration of 0.006% of GE (w/w). This suggests that the combination of these molecules may act in a synergistic manner compared to a single compound. Previously, the anti-fibrotic effects of garlic [26], GEs [17] and some garlic constituents [27] in different models of liver damage have been demonstrated by their antioxidant activity. Indeed, this is the first study to show that the prevention of liver injury by GE may be correlated to the direct inhibition of TG activity that in turn, reduces the levels of enzyme gene expression and transduction, possibly through a mechanism of negative feedback. It is known that in liver tissue TGF- stimulates the hepatic stellate cell differentiation into myofibroblasts that release collagen into the ECM where it is cross-linked by tTG. Furthermore, tTG activates latent TGF-, leading to de novo synthesis of tTG in a self-maintaining system. Data obtained from this study allows us
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to hypothesise that this mechanism, which leads to the formation of fibrosis, can be inhibited by garlic through the blockage of tTG activity and expression. The immunohistochemical study provided further insight into the role of tTG in fibrosis, showing that the enzyme localisation in damaged liver tissue acquires structural morphologies quite similar to that obtained by collagen deposition. Both tTG and collagen staining were strongly reduced by either CYS or GE treatment. This result was consistent with the finding of a down-regulation of alpha-SMA gene expression further supporting the antifibrogenic effect of GE, while the reduced IL-1 expression provides evidence for a role of GE also in reducing inflammation. Several recent studies reported health benefits of garlic diallylsulphides in many pathologies, such as cancer and CVD [28–30]. This study showed the ability of GE in reducing liver fibrosis and strengthening the therapeutic potential of the diallyldisulphide compound and allylmercaptan, which represents the main in vivo metabolite of organosulphur compounds [31]. Today, there is great public interest in natural remedies and in non-conventional therapies for the treatment of several diseases. Evidence supports the use of phytochemicals in combination with conventional therapies to reduce the side effects of pharmaceutical products, making garlic a potential candidate in this scenario. In spite of the numerous studies dealing with the beneficial effects of garlic consumption in humans, none had reported its ability in inhibiting tTG activity before our study. In conclusion, the data shown in this paper suggest that the inhibition of tTG activity obtained by a tailored GE preparation may allow recovery from liver injury and may block the progression of liver fibrosis. Since current therapeutic approaches in treating hepatic fibrosis are limited, a comprehensive understanding of the molecular mechanisms involved in tTG function and its inhibition would provide new therapeutic targets to inhibit the progression of this severe disease. Conflict of interest statement None declared. Acknowledgement D.C. Amoruso was recipient of a SIGE award, year 2008. This work was in part supported by a Campania Municipality grant on functional foods to VF. References [1] Török NJ. Recent advances in the pathogenesis and diagnosis of liver fibrosis. J Gastroenterol 2008;43:315–21. [2] Wanless IR, Nakashima E, Sherman M. Regression of human cirrhosis. Morphologic features and the genesis of incomplete septal cirrhosis. Arch Pathol Lab Med 2000;124:1599–607. [3] Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000;46:443–6. [4] Iredale JP, Benyon RC, Pickering J, et al. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest 1998;102:538–49.
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