Article
L-Theanine
prevents carbon tetrachloride-induced liver fibrosis via inhibition of nuclear factor B and down-regulation of transforming growth factor and connective tissue growth factor
Human and Experimental Toxicology 1–12 ª The Author(s) 2015 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327115578864 het.sagepub.com
JE Pe´rez-Vargas1, N Zarco2, P Vergara2, M Shibayama3, J Segovia2, V Tsutsumi3 and P Muriel1
Abstract Here we evaluated the ability of L-theanine in preventing experimental hepatic cirrhosis and investigated the roles of nuclear factor-B (NF-B) activation as well as transforming growth factor (TGF- ) and connective tissue growth factor (CTGF) regulation. Experimental hepatic cirrhosis was established by the administration of carbon tetrachloride (CCl4) to rats (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks), and at the same time, adding L-theanine (8.0 mg/kg) to the drinking water. Rats had ad libitum access to water and food throughout the treatment period. CCl4 treatment promoted NF-B activation and increased the expression of both TGF- and CTGF. CCl4 increased the serum activities of alanine aminotransferase and g-glutamyl transpeptidase and the degree of lipid peroxidation, and it also induced a decrease in the glutathione and glutathione disulfide ratio. L-Theanine prevented increased expression of NF-B and down-regulated the proinflammatory (interleukin (IL)-1 and IL-6) and profibrotic (TGF- and CTGF) cytokines. Furthermore, the levels of messenger RNA encoding these proteins decreased in agreement with the expression levels. L-Theanine promoted the expression of the anti-inflammatory cytokine IL-10 and the fibrolytic enzyme metalloproteinase-13. Liver hydroxyproline contents and histopathological analysis demonstrated the antifibrotic effect of L-theanine. In conclusion, L-theanine prevents CCl4-induced experimental hepatic cirrhosis in rats by blocking the main pro-inflammatory and pro-fibrogenic signals. Keywords L-Theanine,
liver damage, necrosis, oxidative stress, NF-B, cytokines, cirrhosis, carbon tetrachloride
Introduction Cirrhosis is one of the most frequent and serious liver diseases and is the end stage of progressive fibrosis, which causes major disruptions to metabolic function and blood circulation.1 Cirrhosis is characterized by the accumulation of extracellular matrix (ECM) proteins, and it causes the perturbation of liver homeostasis, leading to the intracellular release of cytokines. In particular, the fibrogenic cytokine, transforming growth factor (TGF- ), is a key factor in cirrhosis development as it stimulates the generation of ECM and inhibits matrix protein removal.
1
Department of Pharmacology, Cinvestav-IPN Apartado, D.F. Me´xico 2 Department of Physiology, Biophysics and Neurosciences, Cinvestav-IPN Apartado, D.F. Me´xico 3 Department of Infectomics and Molecular Pathogenesis, Cinvestav-IPN Apartado, D.F. Me´xico Corresponding author: P Muriel, Department of Pharmacology, Cinvestav-IPN, Apdo. Postal 14–740, Me´xico 07000, D.F. Me´xico. Email: [email protected]
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
2
Human and Experimental Toxicology
Excessive activation of cellular signalling pathways by nuclear factor B (NF-B) has important roles in innate immunity, liver inflammation, fibrosis and the prevention of apoptosis. NF-B constitutes a critical target for manipulating pathophysiological processes in different hepatic diseases. The activation of NF-B is related to oxidative stress-induced cell death. L-Theanine is the primary amino acid in green tea and accounts for 1–2% of the leaf dry weight. There are two isomers of L-theanine, and the L isomer is the predominant form. Its systematic IUPAC name is 2-amino-4-(ethylcarbamoyl) butyric acid and its chemical structure is shown in Figure 1. The structure of L-theanine is very similar to that of glutamic acid, which is a precursor of the main endogenous antioxidant glutathione. L-Theanine is synthesized in the roots of the green tea plant and is concentrated in the leaves.2,3 Previous studies have shown that L-theanine protects cells from damage through its antioxidant activity4 that maintains normal cellular levels of GSH,1 protecting against cancer5,6 and neurotoxicity.7 These observations prompted us to investigate whether L-theanine could play a protective role in cirrhosis induced in a murine experimental model.
Materials and methods Chemicals L-Theanine,
carboxymethylcellulose, thiobarbituric acid, bovine serum albumin, g-glutamyl-p-nitroanilide, L-g-glutamyl-p-nitroaniline, chloramine-T, p-dimethylaminobenzaldehyde and thioacetamide were purchased from Sigma Chemical Company (St Louis, Missouri, USA). Carbon tetrachloride (CCl4), sodium acetate, sodium hydroxide, glacial acetic acid, hydrochloric acid, ethanol, methanol and formaldehyde were obtained from J.T. Baker (Xalostoc, Mexico). All reagents were of analytical quality.
Figure 1. Chemical structure of L-theanine.
(NOM-062-ZOO-1999) and the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985).
Cirrhosis induction Rats initially weighing 100–110 g were divided into four groups of eight rats each. Animals in the control group were given mineral oil (0.25 mL, intraperitoneally (i.p.)) three times per week for 8 weeks. Cirrhosis was induced in the CCl4 group by the administration of CCl4 (0.4 g/kg, i.p.) three times per week for 8 weeks.8,9 The CCl4-L-theanine-treated (THE) group rats were treated as in group CCl4, and they also received daily doses of L-theanine (8.0 mg/kg). Since gavage administration induce stress in animals, and drinking water is an administration route more similar to the human condition, where L-theanine was first consumed as an infusion, and then dissolved in drinking water. Water consumption was measured daily to warrant dosage. The usual dose given to humans in drinking water was 8.0 mg/kg. A dose–response study was not necessary since the dose chosen was effective and safe.The THE group rats received L-theanine only as described for group III. CCl4 and mineral oil were administered in the morning. Animals were killed by exsanguination 72 h following the last dose of CCl4 or mineral oil.
Treatment of animals In this work, male Wistar rats (3 weeks old at the beginning of treatments) were used and were maintained on a standard rat chow diet with free access to drinking water. Eight animals were housed per polycarbonate cage and kept under controlled conditions at 22 + 2 C with 50–60% relative humidity and 12-h light/12-h dark cycles. All animals received humane care, and the study complied with the institution’s guidelines, official Mexican regulations
Biochemical assays Blood sample was collected by cardiac puncture, and the liver was rapidly removed. Serum was obtained to assess liver damage by measuring the enzymatic activities of alanine aminotransferase (ALT)10 and g-glutamyl transpeptidase (g-GTP).11 Assessment of lipid peroxidation. The extent of lipid peroxidation in liver homogenates was determined by
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
3
quantification of malonyldialdehyde (MDA)12 formation by the thiobarbituric acid method. Total protein contents were determined by the Bradford method, using bovine serum albumin as standard.13 Determinations of liver GSH and GSSG. Glutathione (GSH) and glutathione disulfide (GSSG) levels were quantified as described by Hissin and Hilf.14 GSH and GSSG assays were performed, on the same day when the animals were killed, in 0.1 M sodium phosphate, 0.005 M ethylenediaminetetraacetic acid buffer (pH 8.0) and kept on ice until used. Collagen quantification. Collagen concentrations were determined by measuring the hydroxyproline contents of fresh liver samples digested with hydrochloric acid as previously described.15,16
Histology Liver samples were taken from all animals and fixed with 10% formaldehyde in phosphate-buffered saline for 24 h. Tissue pieces were then washed with tap water, dehydrated in alcohol and embedded in paraffin. Five micrometre-thick sections were mounted on glass slides and covered with silane. Staining was performed using haematoxylin and eosin and Masson’s trichromic stain.
Molecular biology assays Total protein isolation. Trizol1 reagent (Invitrogen™, Carlsbad, California, USA) was used to isolate the total protein content of liver tissue, and the total protein levels were determined by the bicinchoninic acid method. Western blot assays. Samples containing 50 mg total protein were separated on 10% polyacrylamide gels, and the proteins were then transferred onto ImmunoBlot™ polyvinylidene difluoride membranes (BioRad, Hercules, California, USA).17 The blots were subsequently blocked with 5% skim milk and 0.05% Tween-20 for 1 h at room temperature and independently incubated at 4 C overnight with antibodies (Abs) selective against each protein: NF-B (p65), TGF- , Metalloproteinase 13 (MMP-13) and interleukin (IL)-1 Abs (MAB3026, MAB1032, MAB13426 and AB1832, respectively) were from Millipore Corp. (Billerica, Massachusetts, USA). IL-6, IL-10 and connective tissue growth factor (CTGF) Abs (SC-57315, SC-57245 and SC14939, respectively)
were from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). Membranes were then washed and exposed to a secondary peroxidaselabelled Ab (Zymed, San Francisco, California, USA) diluted to 1:1500 in blocking solution for 1 h at room temperature. Blots were then washed and developed using the Western Lightning™ Plus-ECL enhanced chemiluminescence detection system (NEN Life Sciences Products, Elmer LAS Inc., Boston, Massachusetts, USA). After stripping, the blots were incubated with a monoclonal Ab directed against -actin,18 as a control for protein loading, and developed using the Western Lightning Plus-ECL kit. Images were digitally captured using a BioDoc-It imaging system (UVP, Upland, California, USA).19 RT-PCR analysis. The messenger RNA (mRNA) levels of the cytokine TGF- in liver samples were measured using reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was isolated from rat liver tissue using the Trizol reagent (Invitrogen), and the concentration and purity of RNA samples were determined by measuring the A260–A280 nm absorbance ratios. Complementary DNA (cDNA) was obtained using poly dT primed RT (Invitrogen), and Taq polymerase (Invitrogen) was used for the PCR reactions.20 The primer sequences and PCR conditions used to amplify TGF- and CTGF are described in Table 1. As a positive control, -actin cDNA was amplified as previously described. Images were digitally acquired using a BioDoc-It Imaging System (UVP).
Statistical analysis Data are expressed as the mean values + SE. Comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey’s test, using the GraphPad Prism 5.00 software. Differences were considered to be statistically significant when p < 0.05.
Results The enzymatic activities of ALT (a marker of necrosis) and g-GTP were increased approximately 1.5-fold in the group treated with CCl4 for 8 weeks compared with the control group, and administration of L-theanine normalized the activities of both the enzymes (Table 2). Chronic exposure to CCl4 increased fibrosis nearly sixfold when compared with the control. This effect
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
4
Human and Experimental Toxicology
Table 1. Primers used for RT-PCR.a Gene
Gene bank accession number
TCF- NM_021578.2 CTGF NM_022266.2
Prime sequence
Number of cycles
Tm ( C)
Product size (bp)
35
58
190
35
58
196
F: 50 AACCCCCATTGCTGTCCCGT-30 R: 50 TTCAGCCACTGCCGGACAAC-30 F: 50 CAAGGACCGCACAGTGGTT-30 R: 50 AACTCTGCTTCTCCAGCCTGC-30
TGF- : transforming growth factor ; CTGF: connective tissue growth factor; RT-PCR: reverse transcriptase polymerase chain reaction. a Primer sequences specific for the indicated rat genes and the sizes of the expected PCR products are shown.
Table 2. Enzyme activities determined in serum in groups of six rats each.a Parameters ALT (mmol/L/min) g-GTP (mmol/L/min)
Control 27.07 + 2.77 9.98 + 1.45
CCl4 þ L-theanine
CCl4 b
79.52 + 2.29 11.29 + 0.70b
b,c
69.86 + 2.05 7.75 + 0.26b,c
L-Theanine
17.70 + 0.96 8.98 + 0.70
CCl4: carbon tetrachloride; g-GTP: g-glutamyl transpeptidase; ALT: alanine aminotransferase. a Effect of the chronic administration of CCl4 on the activities of ALT and g-GTP in control group, CCl4-treated rats (CCl4), CCl4treated rats plus L-theanine (CCl4 þ THE) and rats administered with L-theanine only (THE). Each result represents the mean value of experiments performed in triplicate assays +SE (n ¼ 6). b Significantly different from control at p < 0.05. c Significantly different from CCl4 group at p < 0.05.
was partially, but significantly, inhibited by L-theanine (Figure 2). Sections of rat livers from the different treatment groups were stained with haematoxylin and eosin and are presented in Figure 3. The control group showed a normal appearance (Figure 3(a)), whilst samples from CCl4-treated group showed pronounced nodular fibrosis of the parenchyma with hypertrophic hepatocytes, steatosis and neoformed acidophilic cells (Figure 3(b)). Liver sections from the CCl4 þ L-theanine-treated group revealed that fibrotic regions were diminished and the number of neoformed cells was increased (figure 3(c)), whilst the control group treated with only L-theanine showed a liver parenchyma with normal appearance (Figure 3(d)). Moreover, Masson’s trichromic staining (Figure 4) revealed that small collagen fibres appeared only in portal areas within the livers of the control group (Figure 4(a)). Liver cirrhotic sections as well as nodular fibrosis surrounding hypertrophic hepatocytes were found in tissue samples from the CCl4-treated group (Figure 4(b)). The CCl4 þ L-theanine-treated group showed a reduced level of fibrous tissue (Figure 4(c)) and presented a normal appearance of the liver parenchyma (Figure 4(d)). Membrane oxidative stress was evaluated by determining liver MDA levels. Lipid peroxidation
Figure 2. Liver collagen as measured by hepatic hydroxyproline content in livers from rats following chronic administration of CCl4 for 8 weeks. Control (CONTROL), CCl4-treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. Data represent the mean values of experiments performed in triplicate +SE (n ¼ 6). ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. CCl4: carbon tetrachloride.
was significantly increased by CCl4 treatment, and this increase was partially prevented by L-theanine (Figure 5).
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
5
Figure 3. Haematoxylin and eosin staining of rat liver samples. (a) Untreated control, (b) CCl4 treated, (c) CCL4 þ Ltheanine treated and (d) theanine treated. Bar scale ¼ 50 mm. H: hepatocytes; NH: neoformed hepatocytes; PS: portal space; CCl4: carbon tetrachloride.
Figure 4. Masson’s trichromic stain of rat liver samples. (a) Untreated control, (b) CCl4 treated, (c) CCL4 þ L-theanine treated and (d) theanine treated. Bar scale ¼ 50 mm. CV: central vein; F: fibrous tissue; H: hepatocytes; NH: neoformed hepatocytes; PS: portal space.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
6
Human and Experimental Toxicology
prevented this effect. The cirrhotic group had significantly increased levels of the active form of MMP-13, and L-theanine further increased it. Administration of L-theanine to normal rats did not produce alterations in the expression of either the latent or active forms of this enzyme. Chronic CCl4 intoxication increased both TGF- and CTGF mRNA levels. Interestingly, co-treatment with L-theanine decreased the levels of mRNA encoding these proteins (Figure 14).
Discussion
Figure 5. Liver lipid peroxidation as determined by MDA contents of samples from control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. Results represent the mean value of experiments performed in triplicate +SE (n ¼ 6). ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. MDA: malondialdehyde; CCl4: carbon tetrachloride.
Cytosolic oxidative stress was evaluated by the quantification of liver GSH levels (Figure 6). Decreases in the GSH levels of the CCl4-treated group were observed, and GSSG levels tended to increase in the CCl4-treated group compared with the control group. The administration of CCl4 increased p65 levels compared with the control group and L-theanine partially inhibited this increase (Figure 7). Concomitant increases in IL-1 and IL-6 protein levels in liver samples obtained from CCl4treated rats compared with the control group are shown in Figures 8 and 9, respectively; furthermore, the levels of these cytokines were reduced by co-administration of L-theanine. In contrast, the production of anti-inflammatory cytokine IL-1019 was increased by CCl4 and was further increased by L-theanine (Figure 10). Following the administration of CCl4 over 8 weeks, significant increases in the expression of TGF- (Figure 11) and CTGF (Figure 12) were detected when compared with the control group, and L-theanine diminished the expression of these profibrotic molecules. The effects of L-theanine on MMP-13 activation were evaluated (Figure 13). The latent form (Figure 13(a)) increased in the group treated chronically with CCl4, whilst L-theanine co-administration partially
In the present study, we demonstrated that L-theanine, an amino acid present exclusively in the tea plant (Camellia sinensis), inhibited the necrosis, fibrosis and cirrhosis induced by chronic CCl4 intoxication. L-Theanine is consumed in Asia as a flavouring and relaxing agent in beverages, foods and dietary supplements. L-Theanine toxicity was evaluated by administration of up to 4000 mg/kg daily as a dietary supplement to male and female rats, and no adverse effects were observed.21 The typical doses administered to animals are 2.0 or 4.0 mg/kg; in this study, we administered 8.0 mg/kg similar to the dose given to humans.22 We hypothesized that L-theanine might protect the liver against CCl4-induced injury and fibrosis in part by attenuating oxidative stress. The GSH/GSSG ratio was statistically decreased in the CCl4-treated group in agreement with previous observations.23 L-Theanine effectively preserved the GSH/GSSG ratio, and as oxidative stress is directly associated with hepatic fibrosis,24 the effect of L-theanine on the GSH/GSSG ratio may explain the observed reduction in collagen accumulation. Randle et al.25 reported acute intoxication with xenobiotics including CCl4, increased glutamate cysteine ligase (GCL) activity, and the ratelimiting enzyme in the GSH biosynthetic pathway. However, free radicals produced by chronic CCl4 intoxication reduced hepatic GSH contents as a result of its metabolism (Figure 6); in fact, oxidative stress, including lipid peroxidation and GSH consumption, is one of the most common mechanisms leading to liver damage.26–29 Although inhibition of NF-B could decrease GSH biosynthesis by regulating GCL expression,30,31 the antioxidant properties of L-theanine2 were capable of preventing GSH oxidation (Figure 6) similar to other
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
7
Figure 6. Reduced GSH and oxidized GSSG, GSH/GSSG ratio and total glutathione (GSH þ GSSG) determined in livers from control (CONTROL), CCl4-treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. Each bar represents the mean value of experiments performed in triplicate +SE (n ¼ 6). ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. CCl4: carbon tetrachloride; GSH: glutathione; GSSH: glutathione disulfide.
natural antioxidants.32 Total glutathione levels (GSH þ GSSG) were decreased as a result of GSH reduction in the CCl4-treated group; however, L-theanine prevented the drop in total glutathione, possibly by inhibiting GSH oxidation. Redox homeostasis following 8 weeks of chronic treatment is likely responsible for maintaining glutathione contents without dramatic alterations. Whole liver tissue was used to measure p65, the main subunit of NF-B RelA.33 As we used a specific Ab recognizing p65 that does not cross-react with NF-B when bound to IB, but does when activated, the p65 increase shown in Figure 7 represents the activation of this transcription factor. The CCl4induced NF-B activation is highly dependent on
oxidative stress and liver inflammation is closely associated with the development of hepatic fibrosis and cirrhosis.34,35 In this study, we showed that Ltheanine significantly reduced the increase in NFB expression. Our results are supported by histopathological assays showing that following CCl4 intoxication, the hepatic parenchyma is distorted and fibrosis increased significantly as assessed by hydroxyproline content and visualized by staining with haematoxylin and eosin and trichromic stain. All of these effects were also partially prevented by L-theanine. As demonstrated biochemically and histologically, L-theanine showed anti-fibrotic activity by significantly decreasing the ECM accumulation.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
8
Human and Experimental Toxicology
Figure 7. Expression of NF-B-p65 as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis, and values were calculated as the ratio of p65/ -actin. Each bar represents the mean values determined from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. CCl4: carbon tetrachloride; NF-B: nuclear factor B.
Figure 9. Expression of IL-6 as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of IL1 / -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4treated rats. IL: interleukin; CCl4: carbon tetrachloride.
Figure 8. Expression of IL-1 as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis, and values were calculated as the ratio of IL-1 / actin. Each bar represents the mean values determined from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4treated rats. IL: interleukin; CCl4: carbon tetrachloride.
Figure 10. Expression of IL-10 as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of IL1 / -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4treated rats. IL: interleukin; CCl4: carbon tetrachloride.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
9
Figure 11. Expression of TGF- as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4-treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of TGF- / -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. TGF- :transforming growth factor ; CCl4: carbon tetrachloride.
Figure 12. Expression of CTGF as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of CTGF/ -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. CTGF: connective transforming growth factor; CCl4: carbon tetrachloride.
Cirrhosis is strongly associated with oxidative stress, chronic inflammation and increased TGF- expression.31,34–39 The fibrogenic cytokine TGF- plays a pivotal role in the activation of hepatic stellate cells to generate myofibroblasts, which in turn increase the production of ECM proteins leading to the progression of fibrosis;37 is a prominent pro-fibrogenic cytokine with antiproliferative effects; and can upregulate the deposition of ECM. In this study, L-theanine inhibited TGF- expression and thereby prevented liver fibrosis. CTGF is a downstream mediator of TGF- signalling in fibroblast cells and is widely thought to promote the development of fibrosis in collaboration with TGF- . The crucial role of CTGF in fibrogenesis was evidenced by a significant upregulation of ECM in fibrotic livers.39–41 In this study, we evaluated CTGF protein and mRNA levels, finding that the L-theanine treatment reduced CTGF expression. Furthermore, we found that Ltheanine prevented the increases in IL-1 and IL6 expression.
Degradation of the interstitial matrix is regulated by inhibiting the synthesis of the metalloproteinases and increasing the production of the tissue inhibitor of metalloproteinases. Nevertheless, our results revealed a moderate presence of activated MMP-13 following 8 weeks of CCl4 treatment and a remarkable increase when CCl4-rats were treated with L-theanine. The induction of MMP-13 to degrade the accumulated ECM in liver fibrosis represents an important mechanism of L-theanine action for improving medical treatments of this pathology.
Conclusions The anti-fibrotic properties of L-theanine are associated with its capacity to decrease TGF- and CTGF levels and induce the expression of the active form of MMP-13. The anti-inflammatory and anti-necrotic properties of L-theanine are associated with its ability to inactivate NF-B, a well-known target for liver injury33 and thus decrease pro-inflammatory cytokines
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
10
Human and Experimental Toxicology
Figure 13. Expression of the latent (a) and active (b) forms of MMP-13 as determined by Western blot analysis of hepatic extracts. A representative Western blot is shown (c). Control (CONTROL), CCl4-treated (CCl4), CCl4 plus L-theaninetreated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of MMP-13/ -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. MMP-13: metalloproteinase-13; CCl4: carbon tetrachloride.
Acknowledgements The authors express their gratitude to Biol. Mario Gil Moreno, Mr Ramo´n Herna´ndez and QFB Silvia Galindo Gomez for their excellent technical assistance and to M.V.Z. Rafael Leyva Mun˜oz, MVZ Benjamin E Chavez and M.V.Z. Ricardo Gaxiola for animal handling and care. The authors also acknowledge the Animal Lab Facility, UPEAL-Cinvestav.. The authors also acknowledge the support from Conacyt-PNPC, 2015. Figure 14. mRNA levels of TGF- and CTGF. RT-PCR products were obtained using specific primers for each gene and -actin as described in the text. Control (CONTROL), CCl4-treated (CCl4), CCl4-treated plus L-theanine (CCl4 þ THE) and L-theanine-treated (THE) rats. mRNA : messenger RNS; TGF- : transforming growth factor ; CTGF: connective tissue growth factor; RT-PCR: reverse transcriptase polymerase chain reaction; CCl4: carbon tetrachloride.
(e.g. IL-1 and IL-6) and increase anti-inflammatory IL-10. Moreover, L-theanine has low toxicity and can thus be used in human trials.
Funding JEP-V was a Conacyt fellow (225157) and JS was partially supported by Conacyt grant 127357.
References 1. Ishibashi H, Nakamura M, Komori A, et al. Liver architecture, cell function, and disease. Semin Immunopathol 2009; 31: 399–409. 2. L-theanine. Monograph. Altern Med Rev 2005; 10: 136–138.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
11
3. Lee JE, Lee BJ, Chung JO, et al. Metabolic unveiling of a diverse range of green tea (Camellia sinesis) metabolites dependent on geography. Food Chem 2015; 174: 492–459. 4. Ross SM. L-theanine (suntheanin): effects on L-theanine, an aminoacid derived from Camellia sinensis (green tea), on stress response parameters. Holist Nurse Pract 2014; 28: 65–68. 5. Zhang G, Ye X, Ji D, et al. Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-B pathways with novel theanine derivatives. Oncotarget 2014; 5: 8528–8543. 6. Conde VR, Alves MG, Oliveira PF, et al. Tea (Camellia sinensis (L.)): a putative agent in bladder carcinoma? Anticancer Agents Med Chem 2014; 15: 26–36. 7. Sumathi T, Shobana C, Thangarajeswari M, et al. Protective effect of L-theanine against aluminium induced neurotoxicity in cerebral cortex, hippocampus and cerebellum of rat brain – histopathological, and biochemical approach. Drug Chem Toxicol 2015; 38: 22–31. 8. Perez Tamayo R. Is cirrhosis of the liver experimentally produced by CCl4 and adequate model of human cirrhosis? Hepatology 1983; 3: 112–120. 9. Muriel P. Experimental models of liver damage. In: Saura CS (ed) Hepatotoxicity from Genomics in vitro and in vivo models. New York: John wiley and Sons LTd, 2007, pp. 119–137. 10. Reitman S and Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28: 56–63 11. Glossmann H and Neville DM. Gamma-glutamyl transferase in kidney brush border membranes. FEBS Lett 1972; 19: 340–344. 12. Ohkawa H, Ohishi N and Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351–358. 13. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254. 14. Hissin PJ and Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 1976; 74: 214–226. 15. Prockop DJ and Udenfriend S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1960; 1: 228–239. 16. Muriel P and Deheza R. Fibrosis and glycogen stores depletion induced by prolonged biliary obstruction in
17.
18.
19. 20.
21.
22.
23.
24. 25.
26.
27.
28.
29. 30.
the rat are ameliorated by metadoxine. Liver Int 2003; 23: 262–268. Goldring JP. Protein quantification methods to determine protein concentration prior to electrophoresis. Methods Mol Biol 2012; 869: 29–35. Segovia J, Vergara P and Brenner M. Differentiationdependent expression of transgenes in engineered astrocyte cell lines. Neurosci Lett 1998; 242: 172–176. Bettinger BT, Gilbert DM and Amberg DC. Actin up in the nucleus. Nat Rev Mol Cell Biol 2004; 5: 410–415. Cortez N, Trejo F, Vergara P, et al. Primary astrocytes retrovirally transduced with a tyrosine hydroxylase transgene driven by a glial-specific promoter elicit behavioral recovery in experimental parkinsonism. J Neurosci Res 2000; 59: 39–46. Borzelleca JF, Peters D and Hall W. A 13-week dietary toxicity and toxicokinetic study with l-theanine in rats. Food Chem Toxicol 2006; 44: 1158–1166. Lyon MR, Kapoor MP and Juneja LR. The effects of L-theanine (Suntheanine(R)) on objective sleep quality in boys with attention deficit hyperactivity disorder (ADHD): a randomized, double-blind, placebocontrolled clinical trial. Altern Med Rev 2011; 16: 348–354. Chavez E, Reyes-Gordillo K, Segovia J, et al. Resveratrol prevents fibrosis, NF-B activation and TGF- increases induced by chronic CCl4 treatment in rats. J Appl Toxicol 2008; 28: 35–43. Poli G and Parola M. Oxidative damage and fibrogenesis. Free Radic Biol Med 1997; 22: 287–305. Randle LE, Goldring CEP, Benson CA, et al. Investigation of the effect of a panel of model hepatotoxins on the Nrf2-Keap1 defence response pathway in CD-1 mice. Toxicology 2008; 243: 249–260. Sugiyama T and Sadzuka Y. Theanine and glutamate transporter inhibitors enhance the antitumor efficacy of chemotherapeutic agents. Biochim Biophys Acta 2003; 1653: 47–59. Sadzuka Y, Sugiyama T, Suzuki T, et al. Enhancement of the activity of doxorubicin by inhibition of glutamate transporter. Toxicol Lett 2001; 123: 159–167. Sugiyama T and Sadzuka Y. Theanine, a specific glutamate derivative in green tea, reduces the adverse reactions of doxorubicin by changing the glutathione level. Cancer Lett 2004; 212: 177–184. Muriel P. Role of free radicals in liver diseases. Hepatol Int 2009; 3: 526–536. Meng Q, Peng Z, Chen L, et al. Nuclear factor-B modulates cellular glutathione and prevents oxidative stress in cancer cells. Cancer Lett 2010; 18: 45–53.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
12
Human and Experimental Toxicology
31. Peng Z, Geh E, Chen L, et al. Inhibitor of B kinase regulates Redox homeostasis by controlling the constitutive levels of glutathione. Mol Pharmacol 2010; 77: 784–792. 32. Reyes-Gordillo K, Segovia J, Shibayama M, et al. Curcumin protects against acute liver damage in the rat by inhibiting NF-kappaB, proinflammatory cytokines production and oxidative stress. Biochim Biophys Acta 2007; 1770: 989–996. 33. Muriel P. NF-kappaB in liver diseases: a target for drug therapy. J Appl Toxicol 2009; 29: 91–100. 34. Marra F. Chemokines in liver inflammation and fibrosis. Front Biosci 2002; 7: 1899–1914. 35. Muriel P. Cytokines and liver diseases In: Saura CS (ed) Hepatotoxicity from Genomics in vitro and in vivo models. New York: John wiley and Sons Ltd, 2007, pp. 371–389. 36. Meyer C, Meindl-Beinker NM and Dooley S. TGF-beta signaling in alcohol induced hepatic injury. Front Biosci 2010; 15: 740–749.
37. Gressner AM and Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 2006; 10: 76–99. 38. Gressner AM, Lahme B, Mannherz HG, et al. TGF-beta-mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes. J Hepatol 1997; 26: 1079–1092. 39. Weng HL, Ciuclan L, Liu Y, et al. Profibrogenic transforming growth factor-beta/activin receptor-like kinase 5 signaling via connective tissue growth factor expression in hepatocytes. Hepatology 2007; 46: 1257–1270. 40. Gressner OA, Lahme B, Siluschek M, et al. Activation of TGF-beta within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor. J Cell Mol Med 2008; 12: 2717–2730. 41. Hernandez-Gea V and Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol 2011; 6: 425–56.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
L-Theanine
prevents carbon tetrachloride-induced liver fibrosis via inhibition of nuclear factor B and down-regulation of transforming growth factor and connective tissue growth factor
Human and Experimental Toxicology 1–12 ª The Author(s) 2015 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327115578864 het.sagepub.com
JE Pe´rez-Vargas1, N Zarco2, P Vergara2, M Shibayama3, J Segovia2, V Tsutsumi3 and P Muriel1
Abstract Here we evaluated the ability of L-theanine in preventing experimental hepatic cirrhosis and investigated the roles of nuclear factor-B (NF-B) activation as well as transforming growth factor (TGF- ) and connective tissue growth factor (CTGF) regulation. Experimental hepatic cirrhosis was established by the administration of carbon tetrachloride (CCl4) to rats (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks), and at the same time, adding L-theanine (8.0 mg/kg) to the drinking water. Rats had ad libitum access to water and food throughout the treatment period. CCl4 treatment promoted NF-B activation and increased the expression of both TGF- and CTGF. CCl4 increased the serum activities of alanine aminotransferase and g-glutamyl transpeptidase and the degree of lipid peroxidation, and it also induced a decrease in the glutathione and glutathione disulfide ratio. L-Theanine prevented increased expression of NF-B and down-regulated the proinflammatory (interleukin (IL)-1 and IL-6) and profibrotic (TGF- and CTGF) cytokines. Furthermore, the levels of messenger RNA encoding these proteins decreased in agreement with the expression levels. L-Theanine promoted the expression of the anti-inflammatory cytokine IL-10 and the fibrolytic enzyme metalloproteinase-13. Liver hydroxyproline contents and histopathological analysis demonstrated the antifibrotic effect of L-theanine. In conclusion, L-theanine prevents CCl4-induced experimental hepatic cirrhosis in rats by blocking the main pro-inflammatory and pro-fibrogenic signals. Keywords L-Theanine,
liver damage, necrosis, oxidative stress, NF-B, cytokines, cirrhosis, carbon tetrachloride
Introduction Cirrhosis is one of the most frequent and serious liver diseases and is the end stage of progressive fibrosis, which causes major disruptions to metabolic function and blood circulation.1 Cirrhosis is characterized by the accumulation of extracellular matrix (ECM) proteins, and it causes the perturbation of liver homeostasis, leading to the intracellular release of cytokines. In particular, the fibrogenic cytokine, transforming growth factor (TGF- ), is a key factor in cirrhosis development as it stimulates the generation of ECM and inhibits matrix protein removal.
1
Department of Pharmacology, Cinvestav-IPN Apartado, D.F. Me´xico 2 Department of Physiology, Biophysics and Neurosciences, Cinvestav-IPN Apartado, D.F. Me´xico 3 Department of Infectomics and Molecular Pathogenesis, Cinvestav-IPN Apartado, D.F. Me´xico Corresponding author: P Muriel, Department of Pharmacology, Cinvestav-IPN, Apdo. Postal 14–740, Me´xico 07000, D.F. Me´xico. Email: [email protected]
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
2
Human and Experimental Toxicology
Excessive activation of cellular signalling pathways by nuclear factor B (NF-B) has important roles in innate immunity, liver inflammation, fibrosis and the prevention of apoptosis. NF-B constitutes a critical target for manipulating pathophysiological processes in different hepatic diseases. The activation of NF-B is related to oxidative stress-induced cell death. L-Theanine is the primary amino acid in green tea and accounts for 1–2% of the leaf dry weight. There are two isomers of L-theanine, and the L isomer is the predominant form. Its systematic IUPAC name is 2-amino-4-(ethylcarbamoyl) butyric acid and its chemical structure is shown in Figure 1. The structure of L-theanine is very similar to that of glutamic acid, which is a precursor of the main endogenous antioxidant glutathione. L-Theanine is synthesized in the roots of the green tea plant and is concentrated in the leaves.2,3 Previous studies have shown that L-theanine protects cells from damage through its antioxidant activity4 that maintains normal cellular levels of GSH,1 protecting against cancer5,6 and neurotoxicity.7 These observations prompted us to investigate whether L-theanine could play a protective role in cirrhosis induced in a murine experimental model.
Materials and methods Chemicals L-Theanine,
carboxymethylcellulose, thiobarbituric acid, bovine serum albumin, g-glutamyl-p-nitroanilide, L-g-glutamyl-p-nitroaniline, chloramine-T, p-dimethylaminobenzaldehyde and thioacetamide were purchased from Sigma Chemical Company (St Louis, Missouri, USA). Carbon tetrachloride (CCl4), sodium acetate, sodium hydroxide, glacial acetic acid, hydrochloric acid, ethanol, methanol and formaldehyde were obtained from J.T. Baker (Xalostoc, Mexico). All reagents were of analytical quality.
Figure 1. Chemical structure of L-theanine.
(NOM-062-ZOO-1999) and the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985).
Cirrhosis induction Rats initially weighing 100–110 g were divided into four groups of eight rats each. Animals in the control group were given mineral oil (0.25 mL, intraperitoneally (i.p.)) three times per week for 8 weeks. Cirrhosis was induced in the CCl4 group by the administration of CCl4 (0.4 g/kg, i.p.) three times per week for 8 weeks.8,9 The CCl4-L-theanine-treated (THE) group rats were treated as in group CCl4, and they also received daily doses of L-theanine (8.0 mg/kg). Since gavage administration induce stress in animals, and drinking water is an administration route more similar to the human condition, where L-theanine was first consumed as an infusion, and then dissolved in drinking water. Water consumption was measured daily to warrant dosage. The usual dose given to humans in drinking water was 8.0 mg/kg. A dose–response study was not necessary since the dose chosen was effective and safe.The THE group rats received L-theanine only as described for group III. CCl4 and mineral oil were administered in the morning. Animals were killed by exsanguination 72 h following the last dose of CCl4 or mineral oil.
Treatment of animals In this work, male Wistar rats (3 weeks old at the beginning of treatments) were used and were maintained on a standard rat chow diet with free access to drinking water. Eight animals were housed per polycarbonate cage and kept under controlled conditions at 22 + 2 C with 50–60% relative humidity and 12-h light/12-h dark cycles. All animals received humane care, and the study complied with the institution’s guidelines, official Mexican regulations
Biochemical assays Blood sample was collected by cardiac puncture, and the liver was rapidly removed. Serum was obtained to assess liver damage by measuring the enzymatic activities of alanine aminotransferase (ALT)10 and g-glutamyl transpeptidase (g-GTP).11 Assessment of lipid peroxidation. The extent of lipid peroxidation in liver homogenates was determined by
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
3
quantification of malonyldialdehyde (MDA)12 formation by the thiobarbituric acid method. Total protein contents were determined by the Bradford method, using bovine serum albumin as standard.13 Determinations of liver GSH and GSSG. Glutathione (GSH) and glutathione disulfide (GSSG) levels were quantified as described by Hissin and Hilf.14 GSH and GSSG assays were performed, on the same day when the animals were killed, in 0.1 M sodium phosphate, 0.005 M ethylenediaminetetraacetic acid buffer (pH 8.0) and kept on ice until used. Collagen quantification. Collagen concentrations were determined by measuring the hydroxyproline contents of fresh liver samples digested with hydrochloric acid as previously described.15,16
Histology Liver samples were taken from all animals and fixed with 10% formaldehyde in phosphate-buffered saline for 24 h. Tissue pieces were then washed with tap water, dehydrated in alcohol and embedded in paraffin. Five micrometre-thick sections were mounted on glass slides and covered with silane. Staining was performed using haematoxylin and eosin and Masson’s trichromic stain.
Molecular biology assays Total protein isolation. Trizol1 reagent (Invitrogen™, Carlsbad, California, USA) was used to isolate the total protein content of liver tissue, and the total protein levels were determined by the bicinchoninic acid method. Western blot assays. Samples containing 50 mg total protein were separated on 10% polyacrylamide gels, and the proteins were then transferred onto ImmunoBlot™ polyvinylidene difluoride membranes (BioRad, Hercules, California, USA).17 The blots were subsequently blocked with 5% skim milk and 0.05% Tween-20 for 1 h at room temperature and independently incubated at 4 C overnight with antibodies (Abs) selective against each protein: NF-B (p65), TGF- , Metalloproteinase 13 (MMP-13) and interleukin (IL)-1 Abs (MAB3026, MAB1032, MAB13426 and AB1832, respectively) were from Millipore Corp. (Billerica, Massachusetts, USA). IL-6, IL-10 and connective tissue growth factor (CTGF) Abs (SC-57315, SC-57245 and SC14939, respectively)
were from Santa Cruz Biotechnology Inc. (Santa Cruz, California, USA). Membranes were then washed and exposed to a secondary peroxidaselabelled Ab (Zymed, San Francisco, California, USA) diluted to 1:1500 in blocking solution for 1 h at room temperature. Blots were then washed and developed using the Western Lightning™ Plus-ECL enhanced chemiluminescence detection system (NEN Life Sciences Products, Elmer LAS Inc., Boston, Massachusetts, USA). After stripping, the blots were incubated with a monoclonal Ab directed against -actin,18 as a control for protein loading, and developed using the Western Lightning Plus-ECL kit. Images were digitally captured using a BioDoc-It imaging system (UVP, Upland, California, USA).19 RT-PCR analysis. The messenger RNA (mRNA) levels of the cytokine TGF- in liver samples were measured using reverse transcriptase polymerase chain reaction (RT-PCR). Total RNA was isolated from rat liver tissue using the Trizol reagent (Invitrogen), and the concentration and purity of RNA samples were determined by measuring the A260–A280 nm absorbance ratios. Complementary DNA (cDNA) was obtained using poly dT primed RT (Invitrogen), and Taq polymerase (Invitrogen) was used for the PCR reactions.20 The primer sequences and PCR conditions used to amplify TGF- and CTGF are described in Table 1. As a positive control, -actin cDNA was amplified as previously described. Images were digitally acquired using a BioDoc-It Imaging System (UVP).
Statistical analysis Data are expressed as the mean values + SE. Comparisons were performed using one-way analysis of variance (ANOVA), followed by Tukey’s test, using the GraphPad Prism 5.00 software. Differences were considered to be statistically significant when p < 0.05.
Results The enzymatic activities of ALT (a marker of necrosis) and g-GTP were increased approximately 1.5-fold in the group treated with CCl4 for 8 weeks compared with the control group, and administration of L-theanine normalized the activities of both the enzymes (Table 2). Chronic exposure to CCl4 increased fibrosis nearly sixfold when compared with the control. This effect
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
4
Human and Experimental Toxicology
Table 1. Primers used for RT-PCR.a Gene
Gene bank accession number
TCF- NM_021578.2 CTGF NM_022266.2
Prime sequence
Number of cycles
Tm ( C)
Product size (bp)
35
58
190
35
58
196
F: 50 AACCCCCATTGCTGTCCCGT-30 R: 50 TTCAGCCACTGCCGGACAAC-30 F: 50 CAAGGACCGCACAGTGGTT-30 R: 50 AACTCTGCTTCTCCAGCCTGC-30
TGF- : transforming growth factor ; CTGF: connective tissue growth factor; RT-PCR: reverse transcriptase polymerase chain reaction. a Primer sequences specific for the indicated rat genes and the sizes of the expected PCR products are shown.
Table 2. Enzyme activities determined in serum in groups of six rats each.a Parameters ALT (mmol/L/min) g-GTP (mmol/L/min)
Control 27.07 + 2.77 9.98 + 1.45
CCl4 þ L-theanine
CCl4 b
79.52 + 2.29 11.29 + 0.70b
b,c
69.86 + 2.05 7.75 + 0.26b,c
L-Theanine
17.70 + 0.96 8.98 + 0.70
CCl4: carbon tetrachloride; g-GTP: g-glutamyl transpeptidase; ALT: alanine aminotransferase. a Effect of the chronic administration of CCl4 on the activities of ALT and g-GTP in control group, CCl4-treated rats (CCl4), CCl4treated rats plus L-theanine (CCl4 þ THE) and rats administered with L-theanine only (THE). Each result represents the mean value of experiments performed in triplicate assays +SE (n ¼ 6). b Significantly different from control at p < 0.05. c Significantly different from CCl4 group at p < 0.05.
was partially, but significantly, inhibited by L-theanine (Figure 2). Sections of rat livers from the different treatment groups were stained with haematoxylin and eosin and are presented in Figure 3. The control group showed a normal appearance (Figure 3(a)), whilst samples from CCl4-treated group showed pronounced nodular fibrosis of the parenchyma with hypertrophic hepatocytes, steatosis and neoformed acidophilic cells (Figure 3(b)). Liver sections from the CCl4 þ L-theanine-treated group revealed that fibrotic regions were diminished and the number of neoformed cells was increased (figure 3(c)), whilst the control group treated with only L-theanine showed a liver parenchyma with normal appearance (Figure 3(d)). Moreover, Masson’s trichromic staining (Figure 4) revealed that small collagen fibres appeared only in portal areas within the livers of the control group (Figure 4(a)). Liver cirrhotic sections as well as nodular fibrosis surrounding hypertrophic hepatocytes were found in tissue samples from the CCl4-treated group (Figure 4(b)). The CCl4 þ L-theanine-treated group showed a reduced level of fibrous tissue (Figure 4(c)) and presented a normal appearance of the liver parenchyma (Figure 4(d)). Membrane oxidative stress was evaluated by determining liver MDA levels. Lipid peroxidation
Figure 2. Liver collagen as measured by hepatic hydroxyproline content in livers from rats following chronic administration of CCl4 for 8 weeks. Control (CONTROL), CCl4-treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. Data represent the mean values of experiments performed in triplicate +SE (n ¼ 6). ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. CCl4: carbon tetrachloride.
was significantly increased by CCl4 treatment, and this increase was partially prevented by L-theanine (Figure 5).
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
5
Figure 3. Haematoxylin and eosin staining of rat liver samples. (a) Untreated control, (b) CCl4 treated, (c) CCL4 þ Ltheanine treated and (d) theanine treated. Bar scale ¼ 50 mm. H: hepatocytes; NH: neoformed hepatocytes; PS: portal space; CCl4: carbon tetrachloride.
Figure 4. Masson’s trichromic stain of rat liver samples. (a) Untreated control, (b) CCl4 treated, (c) CCL4 þ L-theanine treated and (d) theanine treated. Bar scale ¼ 50 mm. CV: central vein; F: fibrous tissue; H: hepatocytes; NH: neoformed hepatocytes; PS: portal space.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
6
Human and Experimental Toxicology
prevented this effect. The cirrhotic group had significantly increased levels of the active form of MMP-13, and L-theanine further increased it. Administration of L-theanine to normal rats did not produce alterations in the expression of either the latent or active forms of this enzyme. Chronic CCl4 intoxication increased both TGF- and CTGF mRNA levels. Interestingly, co-treatment with L-theanine decreased the levels of mRNA encoding these proteins (Figure 14).
Discussion
Figure 5. Liver lipid peroxidation as determined by MDA contents of samples from control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. Results represent the mean value of experiments performed in triplicate +SE (n ¼ 6). ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. MDA: malondialdehyde; CCl4: carbon tetrachloride.
Cytosolic oxidative stress was evaluated by the quantification of liver GSH levels (Figure 6). Decreases in the GSH levels of the CCl4-treated group were observed, and GSSG levels tended to increase in the CCl4-treated group compared with the control group. The administration of CCl4 increased p65 levels compared with the control group and L-theanine partially inhibited this increase (Figure 7). Concomitant increases in IL-1 and IL-6 protein levels in liver samples obtained from CCl4treated rats compared with the control group are shown in Figures 8 and 9, respectively; furthermore, the levels of these cytokines were reduced by co-administration of L-theanine. In contrast, the production of anti-inflammatory cytokine IL-1019 was increased by CCl4 and was further increased by L-theanine (Figure 10). Following the administration of CCl4 over 8 weeks, significant increases in the expression of TGF- (Figure 11) and CTGF (Figure 12) were detected when compared with the control group, and L-theanine diminished the expression of these profibrotic molecules. The effects of L-theanine on MMP-13 activation were evaluated (Figure 13). The latent form (Figure 13(a)) increased in the group treated chronically with CCl4, whilst L-theanine co-administration partially
In the present study, we demonstrated that L-theanine, an amino acid present exclusively in the tea plant (Camellia sinensis), inhibited the necrosis, fibrosis and cirrhosis induced by chronic CCl4 intoxication. L-Theanine is consumed in Asia as a flavouring and relaxing agent in beverages, foods and dietary supplements. L-Theanine toxicity was evaluated by administration of up to 4000 mg/kg daily as a dietary supplement to male and female rats, and no adverse effects were observed.21 The typical doses administered to animals are 2.0 or 4.0 mg/kg; in this study, we administered 8.0 mg/kg similar to the dose given to humans.22 We hypothesized that L-theanine might protect the liver against CCl4-induced injury and fibrosis in part by attenuating oxidative stress. The GSH/GSSG ratio was statistically decreased in the CCl4-treated group in agreement with previous observations.23 L-Theanine effectively preserved the GSH/GSSG ratio, and as oxidative stress is directly associated with hepatic fibrosis,24 the effect of L-theanine on the GSH/GSSG ratio may explain the observed reduction in collagen accumulation. Randle et al.25 reported acute intoxication with xenobiotics including CCl4, increased glutamate cysteine ligase (GCL) activity, and the ratelimiting enzyme in the GSH biosynthetic pathway. However, free radicals produced by chronic CCl4 intoxication reduced hepatic GSH contents as a result of its metabolism (Figure 6); in fact, oxidative stress, including lipid peroxidation and GSH consumption, is one of the most common mechanisms leading to liver damage.26–29 Although inhibition of NF-B could decrease GSH biosynthesis by regulating GCL expression,30,31 the antioxidant properties of L-theanine2 were capable of preventing GSH oxidation (Figure 6) similar to other
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
7
Figure 6. Reduced GSH and oxidized GSSG, GSH/GSSG ratio and total glutathione (GSH þ GSSG) determined in livers from control (CONTROL), CCl4-treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. Each bar represents the mean value of experiments performed in triplicate +SE (n ¼ 6). ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. CCl4: carbon tetrachloride; GSH: glutathione; GSSH: glutathione disulfide.
natural antioxidants.32 Total glutathione levels (GSH þ GSSG) were decreased as a result of GSH reduction in the CCl4-treated group; however, L-theanine prevented the drop in total glutathione, possibly by inhibiting GSH oxidation. Redox homeostasis following 8 weeks of chronic treatment is likely responsible for maintaining glutathione contents without dramatic alterations. Whole liver tissue was used to measure p65, the main subunit of NF-B RelA.33 As we used a specific Ab recognizing p65 that does not cross-react with NF-B when bound to IB, but does when activated, the p65 increase shown in Figure 7 represents the activation of this transcription factor. The CCl4induced NF-B activation is highly dependent on
oxidative stress and liver inflammation is closely associated with the development of hepatic fibrosis and cirrhosis.34,35 In this study, we showed that Ltheanine significantly reduced the increase in NFB expression. Our results are supported by histopathological assays showing that following CCl4 intoxication, the hepatic parenchyma is distorted and fibrosis increased significantly as assessed by hydroxyproline content and visualized by staining with haematoxylin and eosin and trichromic stain. All of these effects were also partially prevented by L-theanine. As demonstrated biochemically and histologically, L-theanine showed anti-fibrotic activity by significantly decreasing the ECM accumulation.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
8
Human and Experimental Toxicology
Figure 7. Expression of NF-B-p65 as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis, and values were calculated as the ratio of p65/ -actin. Each bar represents the mean values determined from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. CCl4: carbon tetrachloride; NF-B: nuclear factor B.
Figure 9. Expression of IL-6 as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of IL1 / -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4treated rats. IL: interleukin; CCl4: carbon tetrachloride.
Figure 8. Expression of IL-1 as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis, and values were calculated as the ratio of IL-1 / actin. Each bar represents the mean values determined from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4treated rats. IL: interleukin; CCl4: carbon tetrachloride.
Figure 10. Expression of IL-10 as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of IL1 / -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4treated rats. IL: interleukin; CCl4: carbon tetrachloride.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
9
Figure 11. Expression of TGF- as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4-treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of TGF- / -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. TGF- :transforming growth factor ; CCl4: carbon tetrachloride.
Figure 12. Expression of CTGF as determined by Western blot analysis of hepatic extracts. (a) A representative Western blot is shown. (b) Control (CONTROL), CCl4treated (CCl4), CCl4 plus L-theanine-treated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of CTGF/ -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. CTGF: connective transforming growth factor; CCl4: carbon tetrachloride.
Cirrhosis is strongly associated with oxidative stress, chronic inflammation and increased TGF- expression.31,34–39 The fibrogenic cytokine TGF- plays a pivotal role in the activation of hepatic stellate cells to generate myofibroblasts, which in turn increase the production of ECM proteins leading to the progression of fibrosis;37 is a prominent pro-fibrogenic cytokine with antiproliferative effects; and can upregulate the deposition of ECM. In this study, L-theanine inhibited TGF- expression and thereby prevented liver fibrosis. CTGF is a downstream mediator of TGF- signalling in fibroblast cells and is widely thought to promote the development of fibrosis in collaboration with TGF- . The crucial role of CTGF in fibrogenesis was evidenced by a significant upregulation of ECM in fibrotic livers.39–41 In this study, we evaluated CTGF protein and mRNA levels, finding that the L-theanine treatment reduced CTGF expression. Furthermore, we found that Ltheanine prevented the increases in IL-1 and IL6 expression.
Degradation of the interstitial matrix is regulated by inhibiting the synthesis of the metalloproteinases and increasing the production of the tissue inhibitor of metalloproteinases. Nevertheless, our results revealed a moderate presence of activated MMP-13 following 8 weeks of CCl4 treatment and a remarkable increase when CCl4-rats were treated with L-theanine. The induction of MMP-13 to degrade the accumulated ECM in liver fibrosis represents an important mechanism of L-theanine action for improving medical treatments of this pathology.
Conclusions The anti-fibrotic properties of L-theanine are associated with its capacity to decrease TGF- and CTGF levels and induce the expression of the active form of MMP-13. The anti-inflammatory and anti-necrotic properties of L-theanine are associated with its ability to inactivate NF-B, a well-known target for liver injury33 and thus decrease pro-inflammatory cytokines
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
10
Human and Experimental Toxicology
Figure 13. Expression of the latent (a) and active (b) forms of MMP-13 as determined by Western blot analysis of hepatic extracts. A representative Western blot is shown (c). Control (CONTROL), CCl4-treated (CCl4), CCl4 plus L-theaninetreated (CCl4 þ THE) and L-theanine-treated (THE) rats. -actin was used as a control. Signal intensities were determined by densitometric analysis and values were calculated as the ratio of MMP-13/ -actin. Each bar represents the mean values from three rats +SE in triplicate. ap < 0.05: significant difference from the control; bp < 0.05: significant difference from CCl4-treated rats. MMP-13: metalloproteinase-13; CCl4: carbon tetrachloride.
Acknowledgements The authors express their gratitude to Biol. Mario Gil Moreno, Mr Ramo´n Herna´ndez and QFB Silvia Galindo Gomez for their excellent technical assistance and to M.V.Z. Rafael Leyva Mun˜oz, MVZ Benjamin E Chavez and M.V.Z. Ricardo Gaxiola for animal handling and care. The authors also acknowledge the Animal Lab Facility, UPEAL-Cinvestav.. The authors also acknowledge the support from Conacyt-PNPC, 2015. Figure 14. mRNA levels of TGF- and CTGF. RT-PCR products were obtained using specific primers for each gene and -actin as described in the text. Control (CONTROL), CCl4-treated (CCl4), CCl4-treated plus L-theanine (CCl4 þ THE) and L-theanine-treated (THE) rats. mRNA : messenger RNS; TGF- : transforming growth factor ; CTGF: connective tissue growth factor; RT-PCR: reverse transcriptase polymerase chain reaction; CCl4: carbon tetrachloride.
(e.g. IL-1 and IL-6) and increase anti-inflammatory IL-10. Moreover, L-theanine has low toxicity and can thus be used in human trials.
Funding JEP-V was a Conacyt fellow (225157) and JS was partially supported by Conacyt grant 127357.
References 1. Ishibashi H, Nakamura M, Komori A, et al. Liver architecture, cell function, and disease. Semin Immunopathol 2009; 31: 399–409. 2. L-theanine. Monograph. Altern Med Rev 2005; 10: 136–138.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
Pe´rez-Vargas et al.
11
3. Lee JE, Lee BJ, Chung JO, et al. Metabolic unveiling of a diverse range of green tea (Camellia sinesis) metabolites dependent on geography. Food Chem 2015; 174: 492–459. 4. Ross SM. L-theanine (suntheanin): effects on L-theanine, an aminoacid derived from Camellia sinensis (green tea), on stress response parameters. Holist Nurse Pract 2014; 28: 65–68. 5. Zhang G, Ye X, Ji D, et al. Inhibition of lung tumor growth by targeting EGFR/VEGFR-Akt/NF-B pathways with novel theanine derivatives. Oncotarget 2014; 5: 8528–8543. 6. Conde VR, Alves MG, Oliveira PF, et al. Tea (Camellia sinensis (L.)): a putative agent in bladder carcinoma? Anticancer Agents Med Chem 2014; 15: 26–36. 7. Sumathi T, Shobana C, Thangarajeswari M, et al. Protective effect of L-theanine against aluminium induced neurotoxicity in cerebral cortex, hippocampus and cerebellum of rat brain – histopathological, and biochemical approach. Drug Chem Toxicol 2015; 38: 22–31. 8. Perez Tamayo R. Is cirrhosis of the liver experimentally produced by CCl4 and adequate model of human cirrhosis? Hepatology 1983; 3: 112–120. 9. Muriel P. Experimental models of liver damage. In: Saura CS (ed) Hepatotoxicity from Genomics in vitro and in vivo models. New York: John wiley and Sons LTd, 2007, pp. 119–137. 10. Reitman S and Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin Pathol 1957; 28: 56–63 11. Glossmann H and Neville DM. Gamma-glutamyl transferase in kidney brush border membranes. FEBS Lett 1972; 19: 340–344. 12. Ohkawa H, Ohishi N and Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351–358. 13. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248–254. 14. Hissin PJ and Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem 1976; 74: 214–226. 15. Prockop DJ and Udenfriend S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1960; 1: 228–239. 16. Muriel P and Deheza R. Fibrosis and glycogen stores depletion induced by prolonged biliary obstruction in
17.
18.
19. 20.
21.
22.
23.
24. 25.
26.
27.
28.
29. 30.
the rat are ameliorated by metadoxine. Liver Int 2003; 23: 262–268. Goldring JP. Protein quantification methods to determine protein concentration prior to electrophoresis. Methods Mol Biol 2012; 869: 29–35. Segovia J, Vergara P and Brenner M. Differentiationdependent expression of transgenes in engineered astrocyte cell lines. Neurosci Lett 1998; 242: 172–176. Bettinger BT, Gilbert DM and Amberg DC. Actin up in the nucleus. Nat Rev Mol Cell Biol 2004; 5: 410–415. Cortez N, Trejo F, Vergara P, et al. Primary astrocytes retrovirally transduced with a tyrosine hydroxylase transgene driven by a glial-specific promoter elicit behavioral recovery in experimental parkinsonism. J Neurosci Res 2000; 59: 39–46. Borzelleca JF, Peters D and Hall W. A 13-week dietary toxicity and toxicokinetic study with l-theanine in rats. Food Chem Toxicol 2006; 44: 1158–1166. Lyon MR, Kapoor MP and Juneja LR. The effects of L-theanine (Suntheanine(R)) on objective sleep quality in boys with attention deficit hyperactivity disorder (ADHD): a randomized, double-blind, placebocontrolled clinical trial. Altern Med Rev 2011; 16: 348–354. Chavez E, Reyes-Gordillo K, Segovia J, et al. Resveratrol prevents fibrosis, NF-B activation and TGF- increases induced by chronic CCl4 treatment in rats. J Appl Toxicol 2008; 28: 35–43. Poli G and Parola M. Oxidative damage and fibrogenesis. Free Radic Biol Med 1997; 22: 287–305. Randle LE, Goldring CEP, Benson CA, et al. Investigation of the effect of a panel of model hepatotoxins on the Nrf2-Keap1 defence response pathway in CD-1 mice. Toxicology 2008; 243: 249–260. Sugiyama T and Sadzuka Y. Theanine and glutamate transporter inhibitors enhance the antitumor efficacy of chemotherapeutic agents. Biochim Biophys Acta 2003; 1653: 47–59. Sadzuka Y, Sugiyama T, Suzuki T, et al. Enhancement of the activity of doxorubicin by inhibition of glutamate transporter. Toxicol Lett 2001; 123: 159–167. Sugiyama T and Sadzuka Y. Theanine, a specific glutamate derivative in green tea, reduces the adverse reactions of doxorubicin by changing the glutathione level. Cancer Lett 2004; 212: 177–184. Muriel P. Role of free radicals in liver diseases. Hepatol Int 2009; 3: 526–536. Meng Q, Peng Z, Chen L, et al. Nuclear factor-B modulates cellular glutathione and prevents oxidative stress in cancer cells. Cancer Lett 2010; 18: 45–53.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
12
Human and Experimental Toxicology
31. Peng Z, Geh E, Chen L, et al. Inhibitor of B kinase regulates Redox homeostasis by controlling the constitutive levels of glutathione. Mol Pharmacol 2010; 77: 784–792. 32. Reyes-Gordillo K, Segovia J, Shibayama M, et al. Curcumin protects against acute liver damage in the rat by inhibiting NF-kappaB, proinflammatory cytokines production and oxidative stress. Biochim Biophys Acta 2007; 1770: 989–996. 33. Muriel P. NF-kappaB in liver diseases: a target for drug therapy. J Appl Toxicol 2009; 29: 91–100. 34. Marra F. Chemokines in liver inflammation and fibrosis. Front Biosci 2002; 7: 1899–1914. 35. Muriel P. Cytokines and liver diseases In: Saura CS (ed) Hepatotoxicity from Genomics in vitro and in vivo models. New York: John wiley and Sons Ltd, 2007, pp. 371–389. 36. Meyer C, Meindl-Beinker NM and Dooley S. TGF-beta signaling in alcohol induced hepatic injury. Front Biosci 2010; 15: 740–749.
37. Gressner AM and Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 2006; 10: 76–99. 38. Gressner AM, Lahme B, Mannherz HG, et al. TGF-beta-mediated hepatocellular apoptosis by rat and human hepatoma cells and primary rat hepatocytes. J Hepatol 1997; 26: 1079–1092. 39. Weng HL, Ciuclan L, Liu Y, et al. Profibrogenic transforming growth factor-beta/activin receptor-like kinase 5 signaling via connective tissue growth factor expression in hepatocytes. Hepatology 2007; 46: 1257–1270. 40. Gressner OA, Lahme B, Siluschek M, et al. Activation of TGF-beta within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor. J Cell Mol Med 2008; 12: 2717–2730. 41. Hernandez-Gea V and Friedman SL. Pathogenesis of liver fibrosis. Annu Rev Pathol 2011; 6: 425–56.
Downloaded from het.sagepub.com at UCSF LIBRARY & CKM on April 10, 2015
