Protective effects of garcinol on dimethylnitrosamineinduced
liver fibrosis in rats
Journal: Food & Function
Manuscript ID: FO-ART-04-2014-000342.R2
Article Type: Paper
Date Submitted by the Author: n/a
Complete List of Authors: Pan, Min-Hsiung; National Taiwan University, Institution of Food Science
and Technology
Hung, Wei-Lun; Institute of Food Science and Technology, National Taiwan
University,
Tsai, Mei-Ling; Department of Seafood Science,
Sun, Pei-Pei; National Kaohsiung Marine Unversity,
Tsai, Chen-Yu; Institute of Food Science and Technology, National Taiwan
University,
Yang, Chin-Chou; Department of Seafood Science,
Cheng, An-Chin; Department of Nutrition and Health Sciences,
Ho, Chi-Tang; Rutgers University, Food Science
Food & Function
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Page 1 of 34 Food & Function
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Garcinol markedly reduced the DMN-induced liver fibrosis in rats
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Protective effects of garcinol on dimethylnitrosamine-1 induced liver
2 fibrosis in rats
Wei-Lun Hung1, Mei-Ling Tsai2, Pei-Pei Sun2, Chen-Yu Tsai1, Chin-Chou Yang23 ,
Chi-Tang Ho3, An-Chin Cheng4*, Min-Hsiung Pan1, 5* 4
15
Institute of Food Science and Technology, National Taiwan University, Taipei 10617,
6 Taiwan
27
Department of Seafood Science, National Kaohsiung Marine University, Kaohsiung
8 811, Taiwan
39
Department of Food Science, Rutgers University, New Brunswick, New Jersey
10 08901, USA
411
Department of Nutrition and Health Sciences, Chang Jung Christian University,
12 Tainan 71101, Taiwan
513
Department of Medical Research, China Medical University Hospital, China Medical
14 University, Taichung 40402, Taiwan
15
16 *Please send all correspondence to:
17
18 Dr. Min-Hsiung Pan
19 Institute of Food Science and Technology,
20 National Taiwan University
21 No.1, Section 4, Roosevelt Road, Taipei 10617, Taiwan
22 Tel. no. (886)-2-33664133
23 Fax no. (886)-2-33661771
24 E-mail: mhpan@ntu.edu.tw
25
26 Or
27 Dr. An-Chin Cheng
28 Department of Nutrition and Health Sciences, Chang Jung Christian University,
29 Tainan 71101, Taiwan
30 Tel: +886-2-2785926 Ext 3307
31 E-mail: anniecheng@mail.cjcu.edu.tw
32
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33 ABSTRACT
34 Garcinol, a polyisoprenylated benzophenone derivative, mainly isolated from
35 Garcinia indica fruit rind, has been suggested to exhibit many biological benefits
36 including anti-oxidative, anti-inflammatory, and anti-tumor activities. The aim of this
37 study is to evaluate the protective effects of garcinol on dimethylnitrosamine (DMN)38 induced liver fibrosis in rats. The administration of DMN for six consecutive weeks
39 resulted in the decrease of body weights, the elevation of serum aminotransferases, as
40 well as histological lesions in livers. However, oral administration of garcinol
41 remarkably inhibited the elevation of aspartate transaminase (AST) and relieved liver
42 damage induced by DMN. Furthermore, our results revealed that garcinol not only
43 effectively reduced the accumulation of extracellular matrix (ECM) components but
44 also inhibited the expression of α-smooth muscle actin (α-SMA) in livers. Finally
45 theThe expression of transforming growth factor-β1 (TGF-β1) and the
46 phosphorylation of Smad 2 and Smad 3 were also suppressed by garcinol
47 supplementation. In conclusion, our current study suggested that garcinol exerted
48 hepatoprotective and anti-fibrotic effects against DMN-induced liver injury in rats.
49
50 Keywords: Garcinol, liver fibrosis, dimethylnitrosamine, hepatic stellate cells,
51 transforming growth factor-β1, α-smooth muscle actin
52
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53 1. INTRODUCTION
54 Hepatic fibrosis is the crucial characteristic in the development of chronic liver
55 diseases to cirrhosis and hepatocellular carcinoma (HCC), which is one of the most
reported cancer incidences worldwide156 . A variety of pathological factors attribute to
57 chronic hepatic fibrosis, such as chronic hepatitis B or C infections, alcohol abuse,
chemical intoxication, metabolic syndrome and autoimmune disease258 . Hepatic fibrosis
59 is a wound-healing response of the liver to repeated injury in association with the
60 excessive accumulation of extracellular matrix (ECM) components including
61 collagens, proteoglycans and carbohydrates, leading to architectural disorder and
62 functional impairments. In the pathogenesis of chronic liver disease, reactive oxygen
63 species (ROS) produced from damaged hepatocytes and inflammatory cytokines
64 activate Kupffer cells (the resident macrophages in the liver) and stimulate the
recruitment of activated T cells365 . Kupffer cells are the principal cells in the liver for
66 antigen presentation, phagocytosis, and cytokine production. Injured hepatocytes and
67 Kupffer cells release ROS and pro-inflammatory cytokines, such as transforming
68 growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), as well as interleukins (IL)
69 to further stimulate the activation of hepatic stellate cells (HSCs; also known as
perisinusoidal cells, lipocytes, or Ito cells)470 . The activation of HSCs has consistently
71 been shown to play a crucial role in hepatic fibrogenesis. In the normal liver,
72 quiescent HSCs typically locate in the perisinusoidal space of Disse and serve as the
73 major storage site of lipid-soluble vitamin A. During liver injury, HSCs undergo an
74 activation or transdifferentiation process in which the quiescent cells transform into
75 the proliferative, contractible, and alpha-smooth muscle actin (α-SMA)-expressing
76 myofibroblast-like cells that synthesize ECM proteins.
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Many studies have identified that TGF-β is the most 77 important cytokine
78 contributing to the activation of inflammatory cells and stimulating fibrogenesis of
HSCs579 . For TGF-β signaling, binding of the ligand to TGF- β type II receptor leads to
80 recruitment and phosphorylation the Type I receptor into the complex. The activation
81 of the Type 1 receptor further recruits and phosphorylates Smad 2 and Smad 3, so82
called receptor-activated Smads (R-Smads), which follows binding to Smad 4 to form
83 the complex and translocate into the nucleus where it regulates the target genes such
as collagen type I6, 784 . Therefore, the interruption of the pro-fibrotic signaling for
85 reducing hepatic fibrogenesis and elaboration of antioxidant activities for scavenging
86 free radicals such as ROS are both the potential strategies to inhibit the development
87 of hepatic fibrosis.
88 Dimethylnitrosamine (DMN) is a potent carcinogen, mutagen as well as a
hepatotoxin, and its hepatotoxicity has been first reported in 1954889 . DMN is mainly
90 metabolized by a specific cytochrome P450 isozyme called CYP2E1 and its
metabolites induce hepatotoxicity. George et al9 91 reported that the administration of
92 DMN by intraperitoneal injection for three weeks led to liver injury with hepatocyte
93 necrosis, apoptosis and dysplasia, as well as collagen fiber deposition suggesting that
94 the pathological features and abnormalities of DMN-induced liver injury in rats
95 reflect human hepatic fibrosis. Therefore, the DMN-induced liver injury in rats is a
96 better relevant model to study progression from fibrosis to cirrhosis and HCC.
97 Garcinol, a polyisoprenylated benzophenone derivative, is one of the major
98 active compounds isolated from fruit rind of Garcinia indica (also called Kokum)
99 (Figure 1). The rind of Garcinia indica contains 2-3% garcinol (w/w) and has been
used as a food coloring and traditional medicine in India10, 11100 . Many studies have been
101 reported that garcinol possesses many biological benefits, such as antioxidant activity,
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anti-inflammatory activity and anti-cancer activity12-17. The structural 102 features that
103 confer garcinol with potent antioxidant activity are a β-diketone moiety and phenolic
hydroxyl groups. Yamaguchi et al12 104 has demonstrated that garcinol suppressed the
105 formation of superoxide anion as well as, hydroxyl radical, hydroxyl radical as well as
106 methyl radical in different chemical systems indicating that garcinol is a potent free
107 radical scavenger. Moreover, garcinol also exhibitsed anti-inflammatory effects
108 through inhibiting the expression of inducible nitric oxide synthase (iNOS) and
109 cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages and
astrocytes17, 18110 . Our previous study reported that garcinol could effectively inhibit the
111 growth of Hep 3B cells, a human hepatocellar cancer cell without functional p53,
112 through the elevation of DNA damage-inducible gene 153 (GADD153) expression
and Bax/Bcl-2 ratio, as well as the reduction of mitochondrial membrane potential 19113 .
In addition, Sethi et al20 114 have also found that garcinol exerted inhibitory effects not
115 only on the growth of HCC cells by suppression of the nuclear translocation,
116 phosphorylation and acetylation of signal transducer and activator of transcription 3
117 (STAT3) but also on the size of human HCC xenograft tumors in athymic nu/nu mice.
118 A recent study also documented that the administration of garcinol markedly
119 alleviated LPS/D-galactosamine (D-Gal)-mediated liver injury in rats through
suppressing hepatocyte apoptosis21120 .
121 Since liver fibrosis and cirrhosis are the risk factors in the development of HCC,
122 here, we tested the hypothesis that garcinol has potent anti-fibrotic effects in a DMN123
induced fibrosis model in ratsour current study is to investigate whether the
124 administration of garcinol exerts anti-fibrotic effects in DMN-exposed rats. To our
125 knowledge, it is the first time to utilize DMN-induced live injury as an animal model
126 to evaluate the protective effects of garcinol against liver fibrosis. The possible
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mechanisms of the anti-fibrogenetic effects involved in garcinol-127 mediated regulation
128 of TGF-β/Smad signaling pathway were also discussedinvestigated.
129
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2. METHODS 130 AND METERIALS
131 2.1 Reagents and chemicals
Garcinol was isolated from G. indica dried fruit rind16132 . All reagents and chemicals
133 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). N134
Nitrosodimethylamine (dimethyl N-nitrosamine; DMN) was purchased from Wako
135 Pure Chemical industries Ltd. (Osaka, Japan). Beta-actin antibody was purchased
136 from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TGF- β1, p-Smad 2 and p137 Smad 3 antibodies were purchased from Transduction Laboratories (BD, Biosciences,
138 Lexington, KY, USA). Alpha-SMA antibody was obtained from Epitomics, Inc.
139 (Burlingame, CA, USA).
140
141 2.2 Animals and treatment
142 Healthy male Sprague-Dawley rats (200-250 g) were purchased from BioLASCO Co
143 (Taipei, Taiwan). All animal experimental protocols used in this study were approved
144 by Institutional Animal Care and Use Committee of the National Kaohsiung Marine
145 University (IACUC, NKMU). All animals were housed under a controlled
146 environment (25 ± 1 °C, 65 ± 5 % relative humidity, 12-h lighting period, 0700-1900)
147 and provided with a commercial rodent diet (laboratory rodent diet 5001, LabDiet Co,
148 St. Louis, MO, USA) and distilled water ad libitum throughout the study.
149 After a one-week acclimation, animals were randomly divided into four groups
150 (n=8) including: (i) control, (ii) DMN treatment, (iii) DMN + garcinol
151 supplementation (10 mg/kg bw), and (iv) DMN + garcinol supplementation (25
152 mg/kg bw). The DMN-treated animals were administered DMN (10 mg/kg bw) via
153 intraperitoneal injection three times a week (Mon, Wed, and Fri) for six consecutive
154 weeks. The control group was given normal saline. After administration of DMN for
Formatted: Font: Italic
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three weeks, the animals of the DMN + garcinol supplementation 155 groups were fed
156 with either 10 or 25 mg/kg bw/day of garcinol for three consecutive weeks by oral
157 gavage (Figure 2). The control group was fed with distilled water.
158 At termination of this study, all animals were fasted overnight and euthanized by
159 CO2 anesthesia. The blood samples were collected by cardiac puncture and
160 centrifuged at 1200 × g for 10 min to obtain serum. The vital organs including livers,
161 spleens and kidneys were collected, blotted and weighed. A part (1 cm × 1 cm) of the
162 liver tissue from the right lobe was fixed in 10% buffered formalin solution and
163 embedded in paraffin. The paraffin-embedded tissues were stained with either
164 hemaoxylin & eosin (H&E) or Sirius red. A pathologist reviewed the histology of
165 tissue samples. Quantification of Sirius red-positive areas within hepatic parenchyma
166 was performed by computer-assisted color image analysis (Image J). The remaining
167 liver tissues were frozen in liquid nitrogen and then stored at -80 °C.
168
169 2.3 Biochemical analysis of liver function
170 The serum activities of aspartate transaminase (AST), alanine transaminase (ALT),
171 the levels of total triglyceride (TG) and cholesterol (T-chol) were analyzed by
172 enzymatic methods using an automatic blood biochemistry analyzer (Fujifilm Dri173 Chem 3500s; Fujifilm, Kanagawa, Japan).
174
175 2.4 Tissue protein extraction and western blot analysis
176 The protein extraction and western blot analysis were carried out as previously
described22, 23177 . Briefly, liver tissues from each mouse were homogenized individually
178 and total proteins of livers were extracted by using ice-cold lysis buffer (20 mM Tris179 HCl, pH 7.4; 1 mM NaF; 150 mM NaCl; 1 mM ethylene glycol tetraacetic acid; 1
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mM phenylmethanesulphonyl fluoride; 1% NP-40; and 10 μg/180 mL leupeptin). The
181 concentrations of total protein were determined by Bio-Rad protein assay (Bio-Rad
182 Laboratories, Munich, Germany). The protein sample (50 μg) from each mouse was
183 mixed with 5 × sample buffer, following subjected to SDS-polyacrylamide gel and
184 electrotransferred onto immobile polyvinylidene fluoride (PVDF) membranes
185 (Millipore Corp., Bedford, MA). The membranes were blocked by blocking solution
186 and then immunoblotted with primary antibodies against TGF-β-1, α-SMA, phospho187 Smad 2 (p-Smad 2), phospho-Smad 3 (p-Smad 3) and β-actin for 12 h. After washing
188 with TBST buffer solution, the membranes were applied with horseradish peroxidase189
conjugated secondary antibody for 1 h and then visualized by enhanced
190 chemiluminescence agent (ECL; Amersham Corp., Arlington Heights, IL, USA). The
191 densities of the bands were quantified by densitometric scanning (Alliance 4.7,
192 UVItec, Cambridge, UK). The values are presented as the relative levels of TGF-β1,
193 p-Smad 2, p-Smad 3 and α-SMA over the control group.
194
195 2.5 RNA extraction and reverse transcription-polymerase chain reaction (RT196
PCR)
197 Liver tissues from each mouse were homogenized individually and total RNA was
198 extracted using Trizol reagent (Invitrogen, Carsbad, CA, USA). The RNA samples
199 extracted from liver tissues were transcribed into cDNA by SuperScript II Reverse
200 Transcriptase (Invitrogen, Renfrewshire, UK). The reverse transcription reaction was
201 achieved at 42 °C for 60 min and then 72 °C for 15 min in Gene Cycler thermal cycler
202 (Bio-rad Laboratories, Munich, Germany). The thermal cycle conditions were
203 initiated at 95 °C for 1 min, and then 25 cycles of amplification (94 °C for 30 s, 58 °C
204 for 30 s, 72 °C for 30 s), followed by extension at 72 °C for 3 min. The coding
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sequences of primers are: α-SMA, 205 forward primer 5’206 CGCTGAAGTATCCGATAGAACAC-3’, reverse primer 5’207 CAGTTGTACGTCCAGAGGCATA-3’; β-actin, forward primer 5’208 AAGAGAGGCATCCTCACCCT-3’, reverse primer 5’209 TACATGGCTGGGGTGTTGAA-3’. The amplified products were resolved by 2%
210 agarose gel electrophoresis, stained with ethidium bromide, and visualized under
211 ultraviolet light. The values are presented as the relative levels of TGF-β1, p-Smad2,
212 p-Smad3 and α-SMA over the control group.
213
214 2.6 Statistical analysis
215 Values are presented as means ± standard deviations for the number of experiments
216 indicated. Significant differences were statistically detected by a one-way analysis of
217 variance (ANOVA), followed by using Duncan’s test. Results were considered
218 statistically significant when p < 0.05.
219
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220 3. RESULTS
221 3.1 Effects of garcinol on the body weights and relative organ weights of the
222 DMN-treated rats.
223 The body weights of rats during this study are shown in Figure 3. The body weights
224 of DMN-treated rats were significantly lower than the control rats at the end of the
225 study, whereas no significant differences were found among the DMN-treated animals
226 regardless of garcinol supplementation. The relative organ weights are given in Table
227 1. The relative weights of livers and kidneys in the DMN-alone group showed no
228 significant differences when compared with the control group, whereas the relative
229 weight of spleens was significantly higher than the control group. The
230 supplementation of garcinol did not alter the relative weights of livers, kidneys and
231 spleens when compared with DMN-alone group.
232
233 3.2 Effects of garcinol on the serum biochemical parameters in the DMN-treated
234 rats.
235 ALT, AST, TG and T-cho in serum of rats are shown in Table 2. AST and ALT are
236 the well-known indicators of liver injury. The activities of ALT and AST in DMN237
alone group were significantly higher than the control group, whereas the levels of TG
238 and T-cho were significantly lower than those of the control rats. However, the
239 administration of garcinol to DMN-treated rats did not affect the activities of ALT,
240 the levels of TG and T-cho when compared with DMN-alone group, whereas the
241 activity of AST was significantly reduced.
242
243 3.3 Effects of garcinol on the development of liver fibrosis and the regulation of
244 fibrotic-related signaling pathway in the DMN-treated rats.
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Histological examinations of livers in the control and DMN-245 treated rats by H&E
246 staining were shown in Figure 4. No histological abnormalities were observed in the
247 control group, whereas the DMN-alone group exhibited the congestion and
248 destruction of hepatic architecture, massive and severe hepatocyte necrosis, as well as
249 marked mononuclear cell infiltrates. In contrast, these abnormalities and alterations in
250 the livers were reduced by supplementation with garcinol. The extent of liver fibrosis
251 was also documented by Sirius red staining (Figure 5). A marked increase in Sirius
252 red staining (stained in red) was found in the livers of DMN-alone group when
253 compared with the control group. In contrast, the increase in Sirius red staining was
254 markedly reduced after supplementation with garcinol.
255 The hepatic content of TGF-β1, phospho-Smad 2, phospho-Smad 3 as well as α256 SMA were also measured to evaluate the effect of garcinol against DMN-induced
257 liver fibrosis (Figure 6). TGF-β1 is a crucial fibrotic mediator and our results showed
258 that the expression of TGF-β1 in DMN-alone group was significantly higher than the
259 control group. However, the supplementation of garcinol suppressed the expression of
260 TGF-β1. Similarly, the phosphorylation of Smad 2 and Smad 3 significantly increased
261 by DMN treatment, whereas their phosphorylation was suppressed by garcinol
262 supplementation.
263 Activated HSCs are associated with the accumulation of collagens and the
264 expression of α-SMA. Our results indicated that the expression of α-SMA
265 significantly increased in the DMN-alone group, whereas garcinol supplementation
266 reduced its expression. These results were also consistent with the results of RT-PCR
267 analysis of α-SMA (Figure 7).
268 4. DISCUSSION
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Liver fibrosis involves the excessive deposition of ECM proteins, 269 which is a common
characteristic of most types of chronic liver diseases2270 ; the excessive accumulation of
271 ECM proteins leads to disorganization of the normal lobular architecture by forming a
272 fibrous scar, hepatic functional impairment, and development of nodules of
273 regenerating hepatocytes, consequently resulting in cirrhosis. Hepatic fibrosis and
274 cirrhosis are common risk factors in the development of HCC. It is estimated that
275 HCC is not only the fifth most common cancer but also the third most common cause
of cancer-related deaths1, 24276 . As a result, the prevention of hepatic fibrosis and
277 cirrhosis might be an effective strategy to improve the prognosis of chronic liver
278 disease. DMN-induced liver injury is considered as an appropriate preclinical model
279 to investigate the therapeutic effects of a drug against liver fibrosis because the
280 histopathological features and biochemical alterations induced by DMN resemble the
281 development of liver fibrosis in humans. Thus, in our current study, we utilized the
282 DMN-induced liver fibrosis model to mimic human liver fibrosis and investigate the
283 protective effects of garcinol against liver fibrosis.
284 In this study, we found that the administration of DMN to rats for six
285 consecutive weeks resulted in the decrease of body weights, the increases of serum
286 activities of AST and ALT, as well as the accumulation of ECM components in livers
287 (Tables 1, 2 and Figure 5). George et al. discovered that the decrease in body weights
288 of rats and hepatic accumulation of collagen in livers are found in rats after the
administration of DMN for three consecutive weeks by intraperitoneal injection 9289 .
290 However, garcinol supplementation reduced the elevation of AST activity and the
291 accumulation of ECM components when compared with the DMN-alone group.
292 These results suggested that garcinol exhibited protective effects against DMN293
induced hepatotoxicity as well as liver fibrosis. Similarly, histopathological lesions
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including the destruction of hepatic architecture, hepatocyte necrosis, 294 mononuclear
295 cell infiltrates induced by DMN also greatly attenuated by supplementation of
296 garcinol (Figure 4). Most importantly, previous studies indicated that supplementation
297 of with garcinol did not have negative effects to on the livers of rats. After being fed
298 with thea diet containing 0.05 % garcinol for 5 consecutive weeks, the body weights
299 and liver weights of F344 rats showed no significant differences when compared with
the control group25300 . Similarly, dietary administration of the diet containing 500 ppm
301 garcinol (8.3 mg garcinol/rat/day) for 32 weeks did not cause the decreases in body
weights, liver weights, kidney weights and survival rate of F344 rats 26302 . It is
303 noteworthy that intraperitoneal injection with garcinol (10 mg/ kg bw) did not cause
elevation of AST and ALT activities in BALB/c mice 27304 .
305 Garcinol is a polyisoprenylated benzophenone derivative that is mainly derived
306 from Garcinia indica. Many studies demonstrated that both curcumin and
307 tetrahydrocurcumin exhibit potent antioxidant activities against various diseases due
to their β-diketone moieties and hydroxyl groups on the aromatic rings 28308 . Like
309 curcumin and tetrahydrocurcumin, garcinol is also a potent antioxidant because it
310 contains both a phenolic hydroxyl group and a β-diketone moiety. In many studies,
311 the potent antioxidant activity of garcinol has been used to explain its observed
biological benefits29312 . DMN is a well-known carcinogen and previous studies have
313 indicated that the metabolic activation of DMN resulted in oxidative stress, which
314 may be one of key factors to induce the pathological conditions such as hepatocellular
315 necrosis, carcinogenicity and tumor formation. For example, Vendemiale et al
316 indicated that the elevation of oxidative stress has also been implicated in DMNinduced
fibrosis in rats30317 . As a result, liver damage including the elevation of serum
318 activities of AST and ALT, the disruption of hepatic architecture, as well as
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hepatocyte necrosis found in DMN-treated rats are partially associated 319 with the
320 generation of ROS and RNS. Since many studies have demonstrated that garcinol is a
321 potent antioxidant, the hepatoprotective effects of garcinol against DMN-induced
322 liver damage including decreasing AST and ALT levels, as well as reducing
323 hepatocyte necrosis found in our current study may be, at least in part, due to its
324 potent free radical scavenging ability.
325 Kupffer cells, the resident macrophages of liver, are responsible for protecting
326 hepatocytes by removing foreign particles, mainly microorganisms and bacterial
327 endotoxins, from the portal circulation. They play a key role in innate immune
328 responses and host defense through secretion of inflammatory mediators and ROS.
329 Many pieces of evidence from different studies have suggested that Kupfpfer cells
330 may be implicated in the pathogenesis of various liver diseases such as viral hepatitis,
HCC, alcohol-related liver disease, and liver fibrosis3331 . An animal study indicated that
332 HSC activation and the accumulation of collagens induced by DMN were effectively
inhibited by suppression of macrophage infiltration31333 . In response to liver injury,
334 Kupffer cells produce the pro-inflammatory cytokines and growth factors that induce
335 HSC myofibroblastic transformation. Among the various mediators produced from
336 Kupffer cells or apoptotic hepatocytes, TGF-β1 is recognized as a key cytokine to
337 drive HSC transdifferentiation resulting in increased accumulation of collagen. Many
338 studies have documented that TGF-β1 is the main cytokine that drives fibrosis in
339 different animal models, including alcohol-induced liver fibrosis, carbon tetrachloride
(CCl4)-induced fibrosis, as well as DMN-induced liver fibrosis22, 23, 32, 33340 . Some
341 studies revealed that natural occurring antioxidants exhibited their protective activities
against liver fibrosis by inhibition of TGF-β1 expression34-36342 . Similarly, our current
343 study also demonstrated that the expression of TGF-β1 and the phosphorylation of the
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receptor-activated Smads (R-Smads) including Smad 2 and Smad 3 344 were significantly
345 reduced by supplementation of garcinol. These results suggested that the anti-fibrotic
346 effect of garcinol is partially originated fromdue to its inhibitory effects on HSC
347 activation through suppressing TGF-β1 expression and the TGF-β-mediated signaling
348 pathway. Moreira has also suggested that inactivation of HSCs have been identified as
349 a potential therapeutic target in liver fibrosis such as inhibition of TGF-β and
induction of HSC apoptosis4350 .
351 Liver fibrosis is a complicated pathological process in which multiple cells are
352 involved including HSCs, Kupffer cells and hepatocytes. After acute or chronic liver
353 injury, HSCs undergo an activation or transdifferentiation process and become
354 myofibroblast-like cells with several phenotypes, such as increased proliferation,
355 expression of α-SMA and synthesis of collagens. α-SMA is not only a commonly
356 used marker for the early stage of hepatic fibrosis, but also widely recognized as a
reliable indicator of HSC activation4357 . According to the results of histopathological
358 examinations by H&E, Sirius red staining and western blotting analysis, our data
359 revealed that DMN-induced liver injury resulted in hepatic accumulation of ECM and
360 the elevation of α-SMA expression through the activation of HSCs (Figures 5 and 6).
361 Consistent with a role for garcinol in the inactivation of HSC, our results showed that
362 the expression of α-SMA induced by DMN was also suppressed (Figure 6). The
363 mRNA expression of α-SMA suppressed by garcinol was also confirmed by RT-PCR
364 analysis (Figure 7). Thus, our results suggested that the suppression of α-SMA gene
365 expression induced by DMN was at least in part due to the inactivation of HSCs by
366 garcinol through inhibiting TGF-β1 expression.
367
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368 5. CONCLUSION
369 Our current study demonstrated that garcinol exhibited both hepatoprotective and
370 anti-fibrotic effects against DMN-induced liver injury. The mechanism of anti-fibrotic
371 effects by garcinol was associated with inhibiting the expression of TGF-β1 and α372 SMA, as well as the phosphorylation of Smad 2 and Smad 3. In addition, garcinol
373 might also exert beneficial effects on reducing the oxidative stress induced by DMN
374 as a potent antioxidant, which exhibited the protective effects against liver damage.
375 Thus, our findings suggest that garcinol supplementation may serve as a potential
376 therapeutic strategy against liver fibrosis.
377
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378 ABBREVIATIONS
379 ALT Alanine transaminase
380 AST Aspartate transaminase
381 COX-2 Cyclooxygenase-2
382 DMN Dimethylnitrosamine
383 ECM Extracellular matrix
384 HCC Hepatocellular carcinoma
385 HSCs Hepatic stellate cells
386 iNOS Inducible nitric oxide synthase
387 RNS Reactive nitrogen species
388 ROS Reactive oxygen species
389 TGF-β Transforming growth factor-β
390 TNF-α Tumor necrosis factor-α
391 LPS Lipopolysaccharide
392 α-SMA α-Smooth muscle actin
393 STAT3 Signal transducer and activator of transcription 3
394
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395 ACKNOWLEDGMENT
396 This study was supported by the National Science Council NSC100-2313-B-309-003,
397 NSC 101-2628-B-022-001-MY4, 102-2628-B-002-053-MY3, and NTU-103R7777
398
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399
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469 FIGURE CAPTIONS
470 Figure 1. Chemical structure of garcinol
471 Figure 2. Schematic diagram of the experimental procedure. Rats were divided
472 into four groups including (a) control, (b) DMN-alone (10 mg/kg bw), (c) DMN
473 + low dose of garcinol (10 mg/kg bw) and (d) DMN + high dose of garcinol (25
474 mg/kg bw). The DMN-treated animals were administered DMN (10 mg/kg bw)
475 via intraperitoneal injection three times a week (Mon, Wed, and Fri) for six
476 consecutive weeks. The control group was given normal saline. Rats of DMN +
477 garcinol supplementation groups were fed with either 10 or 25 mg/kg bw/day of
478 garcinol for three consecutive weeks by oral gavage. The control group was fed
479 with distilled water. The animals were sacrificed on day 43. Each group consisted
480 of eight rats.
481 Figure 3. Effects of garcinol on the DMN-induced body weight loss in rats. DMN
482 was intraperitoneally given at a dose of 10 mg/kg body weight three days a week
483 for six consecutive weeks to each group except the control group (n=8). DMN
484 (10 mg/kg): DMN-treated rats; DMN (10 mg/kg) + LG (10 mg/kg): DMN-treated
485 rats with supplementation of 10 mg/kg garcinol; DMN (10 mg/kg) + HG (25
486 mg/kg): DMN-treated rats with supplementation of 25 mg/kg garcinol.
487 Figure 4. Effects of garcinol on the DMN-induced histopathological alterations in rats.
488 Representative hematoxylin and eosin-stained sections of livers are shown from
489 control and DMN-treated rats. (A) Control group; (B) DMN (10 mg/kg): DMN490
treated rats; (C) DMN + Garcinol (10 mg/kg): DMN-treated rats with
491 supplementation of 10 mg/kg garcinol; (D) DMN + Garcinol (25 mg/kg): DMN492
treated rats with supplementation of 25 mg/kg garcinol. Non-overlapping liver
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sections of the animal from each group are shown. The 493 arrows indicate
494 inflammatory foci.
495 Figure 5. Effects of garcinol on the DMN-induced deposition of hepatic ECM
496 components in rats. Representative Sirius red-stained sections of livers from
497 control and DMN-treated rats. (A) Control group; (B) DMN (10 mg/ kg): DMN498
treaed rats; (C) DMN + Garcinol (10 mg/kg): DMN-treated rats with
499 supplementation of 10 mg/kg garcinol; (D) DMN + Garcinol (25 mg/kg): DMN500
treated rats with supplementation of 25 mg/kg garcinol. Representative liver
501 sections of the animal from each group are shown. The arrows indicate the area
502 of DMN-induced deposition of hepatic ECM components. Different liver sections
503 of the animal from each group are shown. Quantification of Sirius red-positive
504 areas within hepatic parenchyma was performed by computer-assisted color
505 image analysis (Image J). Values are expressed as mean ± standard deviation
506 (n=8) and analyzed using one-way ANOVA and Duncan’s test. # p < 0.01 versus
507 control. * p < 0.01 versus DMN.
508 Figure 6. Effects of garcinol on the DMN-induced hepatic protein accumulation of
509 TGF-β1, p-Smad2, p-Smad3 and α-SMA in rats. Total liver cell lysates were
510 analyzed for the hepatic protein accumulation of TGF-β1, p-Smad2, p-Smad3 and
511 α-SMA by Western blot analysis. The western blot is a representative of at least
512 three independent experiments. Quantification of TGF- β1, p-Smad 2, p-Smad 3
513 and α-SMA expression was normalized to β-actin using a densitometer.
514 Figure 7. Effects of garcinol on the DMN-induced mRNA expression of α-SMA in
515 rats. Total liver cell lysates were analyzed for mRNA expression of α-SMA by
516 RT-PCR analysis. Quantification of α-SMA expression was normalized to β-actin
517 using a densitometer.
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Table 1. Effects of garcinol on the DMN-induced relative organ weight 518 alterations in
519 rats
Group
Relative organ weight (g/body weight)
Liver Kidney Spleen
Control 3.44±0.41a 0.79±0.08b 0.17±0.01b
DMN (10 mg/kg) 2.71±0.91a 0.96±0.13ab 0.36±0.02a
DMN+LG (10 mg/kg) 2.80±0.58a 0.98±0.14a 0.37±0.10a
DMN+ HG (25 mg/kg) 2.72±0.93a 1.01±0.25a 0.34±0.10a
520 DMN was intraperitoneally given at a dose of 10 mg/kg body weight three days a
521 week for six consecutive weeks to each group except the control group. Data are
522 expressed as mean ± standard deviation (n=8) and analyzed using one-way ANOVA
523 and Duncan’s test. Different letters represent statistically significant differences
524 among treatments (p<0.05).
525
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Table 2. Effects of garcinol on the DMN-induced serum parameter alterations 526 in rats
Group
Parameter
AST (U/L) ALT (U/L) TG (mg/dL) T-cho (mg/dL)
Control 68.4±10.7b 23.4±4.4b 177.9±54.9a 82.0±9.9a
DMN (10 mg/kg) 206.0±69.3a 115.0±50.5a 89.3±26.2b 52.0±12.9b
DMN+LG (10 mg/kg) 105.3±22.0b 75.3±22.9ab 87.0±17.0b 52.2±8.1b
DMN+ HG (25 mg/kg) 92.6±43.3b 64.0±31.8ab 77.0±52.9b 45.2±25.0b
527 DMN was intraperitoneally given at a dose of 10 mg/kg body weight three days a
528 week for six consecutive weeks to each group except the control group. Data are
529 expressed as mean ± standard deviation (n=8) and analyzed using one-way ANOVA
530 and Duncan’s test. Different letters represent statistically significant differences
531 among treatments (p<0.05).
532
533
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534
535 Figure 1
536
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537
538
539
540 Figure 2
541
DMN (10 mg/kg bw, i.p)
0 7 14 21 28 35 42 43 Days
Water (10 ml/kg bw/day, oral)
Garcinol (10 mg/ kg bw/day, oral)
Garcinol (25 mg/ kg bw/day, oral)
Saline (1 ml/kg bw, i.p)
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542
543
544 Figure 3
545
546
547
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548
549 Figure 4.
550
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551
552
553
554 Figure 5.
555
Sirius red-positive parenchyma (%)
0
5
10
15
20
25
Control - 10 25
Garcinol (mg/kg)
DMN (10 mg/kg)
#
*
*
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556
557
558 Figure 6.
559
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560
561
562 Figure 7.
563
564
565
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