Capsaicin Inhibits Dimethylnitrosamine-Induced Hepatic Fibrosis by

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Capsaicin Inhibits Dimethylnitrosamine-Induced Hepatic Fibrosis by Inhibiting the TGF-#1/Smad Pathway via Peroxisome Proliferator-Activated Receptor Gamma Activation Jae Ho Choi, Sun Woo Jin, Chul Yung Choi, Hyung Gyun Kim, Gi Ho Lee, Yong An Kim, Young Chul Chung, and Hye Gwang Jeong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04805 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 20, 2016

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Journal of Agricultural and Food Chemistry 1

1

Capsaicin Inhibits Dimethylnitrosamine-Induced Hepatic Fibrosis by Inhibiting the

2

TGF-β1/Smad

3

Activation

Pathway

via

Peroxisome Proliferator-Activated Receptor

Gamma

4 5

Short title: Capsaicin inhibits DMN-induced hepatic fibrosis

6 †, ¶

†, ¶

‡, ¶

, Hyung Gyun Kim †, Gi Ho Lee †,

7

Jae Ho Choi

8

Yong An Kim †, Young Chul Chung §, Hye Gwang Jeong †,*

, Sun Woo Jin

, Chul Yung Choi

9 10

†

College of Pharmacy, Chungnam National University, Daejeon, Republic of Korea

11

‡

Jeollanamdo Institute of Natural Resources Research, Jeollanamdo, Republic of Korea

12

§

Department of Food Science, International University of Korea, Jinju, Republic of Korea

¶

These authors contributed equally to this work.

13 14 15 16 17 18

* To whom correspondence should be addressed:

19

Hye Gwang Jeong; College of Pharmacy, Chungnam National University, Daejeon 34134,

20

Republic

21

[email protected]

of

Korea,

Tel:

+82-42-821-5936,

Fax:

22 23 24

ACS Paragon Plus Environment

+82-42-825-4936,

E-mail:

Journal of Agricultural and Food Chemistry

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25

ABSTRACT

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Capsaicin (CPS) exerts many pharmacological effects, but any possible influence on liver

27

fibrosis remains unclear. Therefore, we evaluated the inhibitory effects of CPS on

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dimethylnitrosamine (DMN) and TGF-β1-induced liver fibrosis in rats and hepatic stellate

29

cells (HSCs). CPS inhibited DMN-induced hepatotoxicity, NF-κB activation, and collagen

30

accumulation. CPS also suppressed the DMN-induced increases in α-SMA, collagen type I,

31

MMP-2, and TNF-α. In addition, CPS inhibited DMN-induced TGF-β1 expression (from 2.3

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± 0.1 to 1.0 ± 0.1) and Smad2/3 phosphorylation (from 1.5 ± 0.1 to 1.1 ± 0.1; and from 1.6 ±

33

0.1 to 1.1 ± 0.1, respectively), by activating Smad7 expression (from 0.1 ± 0.0 to 0.9 ± 0.1)

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via PPARγ induction (from 0.2 ± 0.0 to 0.8 ± 0.0 ) (p < 0.05). Furthermore, in HSCs, CPS

35

inhibited the TGF-β1-induced increases in α-SMA and collagen type I expression, via PPARγ

36

activation. These results indicate that CPS can ameliorate hepatic fibrosis by inhibiting the

37

TGF-β1/Smad pathway via PPARγ activation.

38 39 40

Keywords: Capsaicin; liver fibrosis; dimethylnitrosamine; PPARγ; hepatic stellate cells

41 42


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Introduction

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The liver is responsible for the biotransformation and detoxification of exogenous and

45

endogenous metabolites and toxicants.

46

environmental toxins is associated with liver damage, ranging from a transient elevation of

47

liver enzymes to life-threatening hepatic inflammation, fibrosis, cirrhosis, and cancer. Liver

48

fibrosis is a dynamic process commonly preceded by chronic inflammation and excessive

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secretion of matrix proteins by hepatic stellate cells. It is a pathophysiological process

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involved in excessive extracellular matrix (ECM) deposition in response to chronic hepatic

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damage and recovery therefrom, such as that occurring in response to the reactive and

52

reparative processes associated with chronic viral hepatitis, alcohol consumption, fat

53

accumulation, and drug abuse.

54

Hepatic stellate cells (HSCs), the main fibrogenic cell type, play an essential role in ECM

55

remodeling. Activated HSCs increase the proliferation of phenotypically transformed

56

fibroblasts, the migration of which is triggered by the binding of fibrogenic transforming

57

growth factor-beta 1 (TGF-β1) to its receptors.

58

subsequently forms a complex with Smad4 and migrates to the nucleus, where it regulates the

59

expression of genes involved in ECM synthesis and deposition.

60

activation is both an important marker of the development of liver fibrosis and a critical

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therapeutic target.

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Peroxisome

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transcription factor that plays pivotal roles in various pathological processes associated with

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inflammation, obesity, and tumorigenesis, and ECM remodeling. PPAR-γ is highly expressed

65

in quiescent HSCs of the normal liver. PPAR-γ activation inhibits the expression of α-SMA

66

and collagen by reducing HSC proliferation during hepatic fibrogenesis.

1,2

However, exposure of the liver to high levels of

3-5

proliferator-activated

6

Constitutively phosphorylated Smad2/3

receptor-gamma

(PPAR-γ)


is

5,7,8

a

Therefore, HSC

ligand-activated

9,10

Therefore,


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PPAR-γ status is a pivotal marker of the success of anti-fibrotic therapy.

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Natural agents exhibiting anti-fibrotic activity provide an effective alternative therapy for

69

treating chronic liver diseases.

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and less toxic hepatoprotective agents from natural sources for use in treating a variety of

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pathologic conditions. Polyphenols are major plant metabolites of foods or nutraceuticals.

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They are diverse in structure, but possess at least one aromatic ring bearing one or more

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hydroxyl groups. Polyphenols reportedly protect against inflammation, obesity, and cancer by

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exerting anti-oxidative effects and scavenging free radicals. Capsaicin (CPS) is a phenolic

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compound of hot red peppers and chili peppers that is consumed worldwide, and has been

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used as a spice, food additive, and drug. It has chronic anti-inflammatory and

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chemopreventative activities in vitro. CPS counters the activities of certain mutagens and

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exerts anticancer effects on breast, prostate, colon, and liver cancer cells. The proposed

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molecular mechanism includes induction of apoptosis via inhibition of translocation by the

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NF-κB and AP-1 signaling pathways. However, despite these known pharmacological

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activities, the effects of CPS on liver fibrosis have received little attention. There is a need to

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develop new and therapeutic agents for liver fibrosis.

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Therefore, we investigated the inhibitory effects of CPS using an animal model of hepatic

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fibrosis induced by dimethylnitrosamine (DMN). Our findings show that CPS inhibits the

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development of hepatic inflammation and fibrosis via PPARγ induction and should thus be

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explored as an alternative therapeutic agent in patients with chronic liver fibrosis.

11,12

There has also been increasing interest in developing new

87 88

Materials and methods

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Chemicals

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Capsaicin and DMN were obtained from Sigma Chemical (St. Louis, MO, USA). The


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following antibodies were obtained for Western blotting; anti-IκBα, anti-NF-κB p65, anti-

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Lamin B1, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-collagen

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type I, anti-PPARγ (Millipore, Merck KGaA, Darmstadt, Germany), anti-Smad7, anti-TGF-

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β1 (Abcam, Cambridge, MA, USA), anti-α-SMA (Dako, Glostrup, Denmark), anti phospho-

95

Smad2, anti-phospho-Smad3 and secondary antibodies coupled with HRP (anti-rabbit or anti-

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mouse IgGs) (Cell Signal Technology Inc., Beverly, MA, USA). Polyvinylidene fluoride

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membranes were purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). The

98

Western blot detection kit was purchased from iNtRON Biotechnology Co., Ltd. (Seoul,

99

Korea).

100 101

Animals and DMN-induced liver injury

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Male Sprague-Dawley rats (130–140 g) were obtained from Samtako (Osan, Korea). Rats

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were acclimatized for at least 2 weeks prior to the experiments and were allowed free access

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to a certified rodent diet (LabDiet 5002, Orient Bio Inc., Gyeonggi-Do, Korea) and tap water.

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All experimental protocols for animal care were performed according to standard guidelines

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and complied with the rules and regulations of the Animal Ethics Committee, Chungnam

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National University. The rats were divided into four groups (n = 5 in each group). To induce

108

hepatic fibrosis, rats were injected intraperitoneally (i.p.) with DMN dissolved in sterile

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saline (10 mg/kg body weight) three times per week for 4 weeks. 13 CPS dissolved in absolute

110

ethanol and then diluted with saline to yield final doses of 0.5 and 1.0 mg/kg was

111

administered to the rats intragastrically (i.g.) six times per week for 4 weeks, with treatment

112

occurring 1 h before DMN administration. Rats in the control and DMN-treated groups were

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administered saline instead of CPS. At the end of the study, all of the animals were fasted

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overnight and euthanized via CO2 anesthesia (Figure 1). Blood was collected and sera



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separated by centrifugation. The livers were collected, blotted, and weighed. Each right lobe

116

(1 cm × 1 cm) was fixed in 10% (v/v) buffered formalin.

117 118

Biochemical analysis and ELISA

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Hepatotoxicity was determined by measuring serum ALT and AST activities using diagnostic

120

kits (Asan Pharmaceutical Co., Seoul, Korea). Lipid peroxidation was analyzed by measuring

121

thiobarbituric acid reactive substances (TBARS). Phosphorylation of NF-κB p65 (Ser536)

122

and IκBα (Ser32) was measured using PathScan Sandwich ELISA kits (Cell Signaling

123

Technology, Beverly, MA, USA). Each absorbance was corrected by reference to that of the

124

negative control and normalized to those of NF-κB p65 and IκBα.

125 126

Histopathological examination and immunohistochemical staining

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Histopathological examinations were carried out as described previously. 14 Histopathological

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changes were examined by light microscopy. Microscopic fields for examination were chosen

129

randomly and viewed at a magnification of ×100. A minimum of 10 fields were scored per

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liver slice. The extent of fibrosis was graded according to Knodell’s scoring method. 15

131 132

Hepatic collagen content

133

Hepatic collagen content was determined using the Sircol collagen assay kit. 14

134 135

HSC cell culture

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HSC-T6, an immortalized rat HSC line, was cultured at 37°C in a 5% (v/v) carbon dioxide

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humidified atmosphere in DMEM media (Gibco BRL, Grand Island, NY) containing 10%

138

(v/v) fetal bovine serum (FBS), streptomycin (100 µg/mL), and penicillin (100 U/mL)


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139 140

Semi-quantitative RT-PCR

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Total RNA was isolated from liver tissue samples using RNAiso reagent (Takara, Kyoto,

142

Japan). After reverse transcription of 0.5 µg RNA, semi-quantitative RT-PCR was performed

143

as described previously.

144

program. The PCR primer sequences are listed in Table S1.

14

RT-PCR bands densities were obtained using NIH Image J

145 146

Western blotting

147

Hepatic protein expression levels were determined by Western blotting.

148

were obtained using NIH Image J software.

14

Band densities

149 150

Statistical analysis

151

The results are reported as the means ± SEMs. All of the data were compared by one-way

152

analysis of variance, followed by Tukey-Kramer multiple comparisons testing. A p-value less

153

than 0.05 indicates statistical significance.

154 155

RESULTS

156

CPS inhibited hepatotoxicity in DMN-treated rats.

157

Chemically induced hepatic damage is related to increased oxidative stress and lipid

158

peroxidation, which can lead to liver dysfunction. 16 Repeated injection of DMN causes liver

159

fibrosis

160

manifestations of liver injury. In rats treated for 4 weeks with DMN, the elevated serum ALT,

161

AST and TBARS levels together with reduction of body and liver weights evidenced

162

hepatotoxicity and hepatic lipid peroxidation. All of these effects were dramatically inhibited

17

and has been traditionally used to study the biochemical and pathological



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by CPS treatment (Table 1).

164 165

CPS inhibited DMN-induced α-SMA and collagen I expression in rats.

166

The transactivation of quiescent HSCs is one of the hallmarks of hepatic fibrosis in the

167

fibrotic liver. Activated HSCs induce the accumulation of collagen and ECM deposition. 18

168

Repeated DMN injection results in severe liver injury with increased collagen synthesis, thick

169

fibrous bands, an inflammatory infiltrate, and hepatocytes injury in the liver, as shown by

170

staining of hematoxylin and eosin (H&E) as well as Masson’s trichrome. Chronic DMN

171

exposure also leads to the increased hepatic deposition of α-SMA, a marker of the

172

transactivation of HSCs to myofibroblasts. CPS treatment dramatically improved the

173

histopathological changes, reduced ECM deposition (Figure 2), and significantly suppressed

174

the expression of α-SMA and collagen I induced by DMN (Figure 3A, B). These results

175

indicate that CPS has the anti-fibrotic effect in rats with DMN-induced liver fibrosis.

176 177

CPS inhibited MMP-2 and TIMP-1 expression in rats with DMN-induced liver fibrosis

178

An imbalance between enhanced ECM synthesis and reduced ECM decomposition is a main

179

cause of liver fibrosis. Matrix metalloproteinase-2 (MMP-2) increases ECM synthesis during

180

hepatic fibrogenesis, but tissue inhibitor of metalloproteinase-1 (TIMP-1) controls the rate of

181

ECM degradation, and is thus an important regulator of liver fibrosis. We evaluated the

182

inhibitory effects of CPS on DMN-induced ECM-associated markers in liver tissue. Repeated

183

DMN injections induced MMP-2 expression and reduced TIMP-1 expression. However, CPS

184

dose-dependently inhibited the DMN-induced MMP-2 expression and the DMN-reduced

185

TIMP-1 expression (Figure 3C, D).

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CPS inhibited DMN-induced TNF-α and NF-κB activation in rats.

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Damaged liver cells secrete cytokines that stimulate inflammation and contribute to the

189

pathogenesis of chronic liver diseases.

190

levels of DMN-induced inflammatory markers in liver tissue. The hepatic level of TNF-α, a

191

cytokine released from Kupffer cells after liver injury, increased in response to repeated

192

DMN injections. CPS treatment dose-dependently inhibited the DMN-induced increase in

193

TNF-α expression (Figure 4A). NF-κB, an important transcription factor, regulates MMP-2

194

and TNF-α expression during liver fibrogenesis. The activation of NF-κB by a variety of

195

extracellular stimuli leads to the phosphorylation, ubiquitination, and finally, proteolytic

196

degradation of the NF-κB inhibitor IκB. The subsequent release of NF-κB from IκB allows it

197

to translocate to the nucleus.

198

phosphorylation, IκBα degradation, and nuclear translocation of NF-κB p65, in a dose-

199

dependent manner (Figure 4B–D). Thus, the inactivation of NF-κB p65 is an additional

200

mechanism by which CPS inhibits liver fibrosis.

20

19

We evaluated the inhibitory effects of CPS on the

CPS inhibited DMN-induced NF-κB p65 and IκBα

201 202

CPS inhibited DMN-induced TGF-β1-dependent Smad regulation in rats.

203

TGF-β1, a potent fibrogenic cytokine, activates HSCs in the liver as part of a spectrum of

204

pathological responses, including the enhancement of α-SMA expression, ECM formation,

205

Smad activation, and collagen expression.

206

DMN-induced increase in TGF-β1 expression as well as Smad2 and Smad3 phosphorylation

207

in liver tissue, in addition to restoring the DMN-induced reduction in hepatic Smad7

208

expression (Figure 5). These results demonstrate the inhibitory effect of CPS on an

209

underlying mechanism of liver fibrosis: TGF-β1-dependent Smad2/3 activation through

210

Smad7 inactivation.

21

CPS treatment dose-dependently inhibited the



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Effects of CPS on PPARγγ expression

213

PPARγ, a group of nuclear receptor isoforms, blocks HSC activation and thereby inhibits

214

hepatic fibrogenesis. Several studies have reported that up-regulation of PPARγ induces an

215

anti-fibrotic effect in mice and rats with CCl4- or DMN-induced liver fibrosis. 13,22 We found

216

that PPARγ expression was reduced by repeated DMN injections but was restored by CPS in

217

a dose-dependent manner (Figure 6). We also confirmed the effect of CPS on TGF-β1-

218

induced reduction in PPARγ expression. We observed no significant differences in HSC-T6

219

cell viability after CPS treatment at any concentration (data not shown). Figure 7A shows that

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TGF-β1 reduced PPARγ expression in a concentration- and time-dependent manner. CPS

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inhibited the TGF-β1-induced reduction in PPARγ expression in a dose-dependent manner.

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Furthermore, a PPARγ antagonist, GW9662, partially reversed this process (Figure 7B). CPS

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also inhibited the TGF-β1-induced increases in α-SMA and collagen type I expression; both

224

of these effects were partially reversed by GW9662 (Figure 7C, D). Thus, by inhibiting liver

225

fibrosis through PPARγ activation, CPS offers a promising therapeutic strategy for patients

226

with liver fibrosis.

227 228

DISCUSSION

229

Liver injury is induced by a wide range of factors, including alcohol abuse, obesity, chemical

230

injection, and hepatitis virus infection. Hepatic fibrosis reflects the cellular and molecular

231

wound-healing responses to liver damage. The latter is characterized by the persistent

232

production of inflammatory cytokines, proteolytic enzymes, angiogenic factors and growth

233

factors, together with enhanced ECM deposition.

234

fibrosis can progress to irreversible hepatic cirrhosis and in the end lead to organ failure

23,24

Without proper treatment, hepatic


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and/or death. Although several studies have reported considerable progress in targeting the

236

pathogenesis of hepatic fibrosis, effective therapeutic agents have yet to be developed.

237

Well-characterized inducers of hepatotoxicity include CCl4, DMN, thioacetamide, ethanol,

238

and bile duct ligation. Chemically induced liver injuries lead to liver dysfunction via the

239

production of ROS. DMN-induced hepatic fibrosis is a useful model of human liver fibrosis

240

because it mimics most of its clinical symptoms, including hepatotoxicity, inflammation, the

241

overproduction of ECM, and the characteristic histopathological changes. 25 Therefore, in this

242

study, we examined the potential therapeutic effects of CPS using DMN-induced hepatic

243

fibrosis as the experimental animal model.

244

Natural agents with good efficacy and a low risk of adverse effects have been used in the

245

prevention and treatment of hepatic inflammation, fibrosis, and cancer. CPS, one of the active,

246

pungent compounds in chili pepper, has and anti-oxidant, anti-inflammatory, and anti-obesity

247

activities.

248

deposition via the inhibition of HSC activation-on hepatic fibrosis induced by bile duct

249

ligation and CCl4 in mice was recently demonstrated.

250

TGF-β effects was not investigated. Therefore, in this study, we evaluated the inhibitory

251

effects of CPS on DMN-induced hepatic fibrosis in rats, in which TGF-β is known to play a

252

role.

253

In the chemically damaged liver, levels of ALT and AST are elevated in serum,

254

generation of ROS causes the oxidation of hepatic lipids. In this study, all three indicators

255

were increased in response to DMN-induced liver fibrosis. Our results showed that CPS

256

administration dose-dependently inhibited serum hepatotoxicity and

257

peroxidation and increased the weights of body and liver in DMN-treated rats. These results

258

are accordance with those in previously reported studies, 28,29 and demonstrate the protective

26,27

Its inhibitory effects-a reduction in α-SMA-positive cells and collagen

27

However, liver fibrosis related to


9

and the

hepatic lipid


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259

effect of CPS in the setting of chronic liver diseases.

260

HSC activation is a pivotal step in the development of hepatic fibrosis. Liver injury increases

261

the phenotypic transformation of HSCs to α-SMA-positive myofibroblasts and stimulates

262

cellular proliferation, the production of inflammatory cytokines, and ECM deposition.

263

Histopathological examination using H&E and Masson’s trichrome staining showed that CPS

264

prevented the extensive changes and collagen deposition in the liver that were induced by

265

DMN. Immunohistochemical staining showed that CPS attenuated the DMN-induced

266

expression of α-SMA-positive cells. Western blotting confirmed the CPS-mediated inhibition

267

of DMN-induced increases in α-SMA and collagen type I. At the mRNA level, CPS also

268

inhibited the DMN-induced MMP-2 expression and DMN reduced TIMP-1 expression.

269

Together, these results demonstrate the inhibitory effect of CPS on HSC activation by DMN.

270

In previous work, we showed that DMN-induced hepatic fibrosis is a chronic inflammatory

271

response to liver injury and that Platycodi Radix, an extract of the flowering plant Platycodon

272

grandiflorum, inhibited hepatic inflammation by reducing NF-κB activation via the induction

273

of antioxidant enzymes. 14 The present study showed that pretreatment with CPS inhibits the

274

DMN-induced increase in NF-κB activation and IκBα degradation, similar to the ability of

275

CPS to suppress PMA- or Helicobacter-pylori-induced NF-κB activation by a similar

276

mechanism. 31,32 We also demonstrated the ability of CPS to inhibit the ROS production, as

277

previously reported in HepG2 cells; in the latter study, the effect was attributed to the

278

increased nuclear translocation of Nrf2 and the expression of HO-1.

279

findings highlight the role of antioxidant enzymes and their inducers, including CPS, in

280

reducing hepatic inflammation and therefore liver fibrosis.

281

TGF-β1, a major fibrogenic cytokine in the liver, stimulates the activation of HSCs by

282

increasing the synthesis and secretion of type-1 collagen to promote ECM formation. The


33

30

Together, these

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283

binding of TGF-β1 to its type II receptor leads to the recruitment and phosphorylation of the

284

type I receptor and its incorporation into the complex. Phosphorylated Smad2/3 binds to

285

Smad4 to form a trimer, which then translocates to the nucleus and binds to the promoter of

286

the TGF-β-responsive element.

287

fibrotic changes in the liver by inhibiting TGF-β-induced Smad2 and Smad3 activation, as

288

previously reported in an experimental animal model of liver fibrosis.

289

administration inhibited DMN-induced increases in TGF-β1 expression and Smad2/3

290

phosphorylation, and maintained Smad7 expression. Our results imply that liver fibrosis can

291

be inhibited, at least partially, by blocking the TGF-β1/Smad signaling pathway, as achieved

292

with CPS (Figure 8).

293

PPARγ plays an important role in the transcriptional regulation of genes involved in

294

adipogenesis, fat deposition, insulin sensitivity, the inflammatory response, and hepatic

295

fibrosis.

296

quiescent, 37 and it has been shown that PPARγ agonists/ligands inhibit both liver fibrosis and

297

HSC activation by activating the PPARγ pathway. 13,38 Previous reports described the anti-

298

fibrogenic effect of curcumin in CCl4-induced liver fibrosis. Curcumin’s mechanism of action

299

includes the attenuation of oxidative stress, suppression of inflammation, inhibition of HSC

300

proliferation, induction of apoptosis, and suppression of ECM production via PPARγ

301

activation.

302

expression, in agreement with the reported ability of CPS to inhibit TGF-β1 secretion, as well

303

as collagen secretion and expression in HSCs by activating PPARγ expression.

304

findings thus demonstrate that CPS is able to prevent DMN-induced liver fibrosis by acting

305

on several of the responsible pathways (Figure 8).

306

A clinical study reported that CPS-induced satiety may be involved in pain from

36

8,34

Smad7 inhibits the trans-differentiation of HSCs and

35

In our study, CPS

In the liver, PPARγ induces a phenotypic switch in HSCs, from activated to

39,40

Our results showed that CPS administration restored DMN-reduced PPARγ


41

Our


Page 14 of 31 14

42

307

gastrointestinal stress,

308

appropriate dosing of CPS for treating of patients with liver fibrosis. Therefore, CPS should

309

be further studied on the proper management and use as a candidate anti-fibrotic agent before

310

the clinical application for patients with liver fibrosis.

and it indicates that there is still an important task to determine

311 312 313

ABBREVIATIONS USED

314

ALT, alanine aminotransferase; α-SMA, alpha-smooth muscle actin; AST, aspartate

315

aminotransferase; CPS, Capsaicin; DMN , dimethylnitrosamine; ECM, extracellular matrix;

316

HSCs, hepatic stellate cells; PPARγ, Peroxisome proliferator-activated receptor-γ; TBARS,

317

thiobarbituric acid reactive substances; TGF-β1, transforming growth factor-beta 1; TIMP-1,

318

tissue inhibitor of metalloproteinase-1; ROS, reactive oxygen species.

319 320 321

ACKNOWLEDGEMENTS

322

This research was supported by Basic Science Research Program through the National

323

Research Foundation of Korea (NRF) funded by the Ministry of Education (2009-0093815),

324

Republic of Korea.

325 326 327

Notes

328

The authors declare no competing financial interest.

329


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Image analysis of dermal collagen changes during skin aging. Analytical and quantitative cytology and histology 1998, 20, 493-9. (16)Zheng, X. Y.; Yang, Y. F.; Li, W.; Zhao, X.; Sun, Y.; Sun, H.; Wang, Y. H.; Pu, X. P., Two xanthones from Swertia punicea with hepatoprotective activities in vitro and in vivo. Journal of ethnopharmacology 2014, 153, 854-63. (17)Guengerich, F. P.; Kim, D. H.; Iwasaki, M., Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. Chemical research in toxicology 1991, 4, 168-79. (18)Suarez-Cuenca, J. A.; Chagoya de Sanchez, V.; Aranda-Fraustro, A.; Sanchez-Sevilla, L.; Martinez-Perez, L.; Hernandez-Munoz, R., Partial hepatectomy-induced regeneration accelerates reversion of liver fibrosis involving participation of hepatic stellate cells. Experimental biology and medicine 2008, 233, 827-39. (19)Tipoe, G. L.; Leung, T. M.; Liong, E. C.; Lau, T. Y.; Fung, M. L.; Nanji, A. A., Epigallocatechin-3-gallate (EGCG) reduces liver inflammation, oxidative stress and fibrosis in carbon tetrachloride (CCl4)-induced liver injury in mice. Toxicology 2010, 273, 45-52. (20)Chen, H. J.; Kang, S. P.; Lee, I. J.; Lin, Y. L., Glycyrrhetinic acid suppressed NFkappaB activation in TNF-alpha-induced hepatocytes. Journal of agricultural and food chemistry 2014, 62, 618-25. (21)Fang, L.; Huang, C.; Meng, X.; Wu, B.; Ma, T.; Liu, X.; Zhu, Q.; Zhan, S.; Li, J., TGF-beta1-elevated TRPM7 channel regulates collagen expression in hepatic stellate cells via TGF-beta1/Smad pathway. Toxicology and applied pharmacology 2014, 280, 335-44. (22)Moran-Salvador, E.; Titos, E.; Rius, B.; Gonzalez-Periz, A.; Garcia-Alonso, V.; Lopez-Vicario, C.; Miquel, R.; Barak, Y.; Arroyo, V.; Claria, J., Cell-specific PPARgamma deficiency establishes anti-inflammatory and anti-fibrogenic properties for this nuclear receptor in non-parenchymal liver cells. Journal of hepatology 2013, 59, 1045-53. (23)Lee, U. E.; Friedman, S. L., Mechanisms of hepatic fibrogenesis. Best practice & research. Clinical gastroenterology 2011, 25, 195-206. (24)Lee, Y.; Friedman, S. L., Fibrosis in the liver: acute protection and chronic disease. Progress in molecular biology and translational science 2010, 97, 151-200. (25)Kao, H. W.; Chen, C. L.; Chang, W. Y.; Chen, J. T.; Lin, W. J.; Liu, R. S.; Wang, H. E., (18)F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model. Bioorganic & medicinal chemistry 2013, 21, 912-21. (26)Cheng, Q.; Li, N.; Chen, M.; Zheng, J.; Qian, Z.; Wang, X.; Huang, C.; Xu, S.; Shi, G., Cyclooxygenase-2 promotes hepatocellular apoptosis by interacting with TNFalpha and IL-6 in the pathogenesis of nonalcoholic steatohepatitis in rats. Dig Dis Sci 2013, 58, 2895-902. (27)Tang, J.; Luo, K.; Li, Y.; Chen, Q.; Tang, D.; Wang, D.; Xiao, J., Capsaicin attenuates LPS-induced inflammatory cytokine production by upregulation of LXRalpha. International immunopharmacology 2015, 28, 264-9. (28)Bitencourt, S.; Stradiot, L.; Verhulst, S.; Thoen, L.; Mannaerts, I.; van Grunsven, L. A., Inhibitory effect of dietary capsaicin on liver fibrosis in mice. Molecular nutrition & food research 2015, 59, 1107-16. (29)Shubha, M. C.; Reddy, R. R.; Srinivasan, K., Antilithogenic influence of dietary capsaicin and curcumin during experimental induction of cholesterol gallstone in mice. Applied physiology, nutrition, and metabolism = Physiologie appliquee,


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473 474

Table 1. Effects of CPS on DMN-induced hepatotoxicity in rats. ALT (IU/L)

AST (IU/L)

TBARS (nmole/g liver)

Control

42.0 ± 1.5

70.7 ± 1.4

3.6 ± 0.4

DMN 10mg/kg

119 ± 4.5

DMN + CPS 0.5 mg/kg

107 ± 9.5

149 ± 16.3

9.0 ± 0.2

DMN + CPS 1.0 mg/kg

85.4 ± 9.7 *

100 ± 9.9 *

5.7 ± 0.9 *

#

166 ± 17.8

#

9.5 ± 0.7

#

475

476

Results are expressed as the means ± SEM. # P < 0.05, significantly different from the control

477

group. * P < 0.05, significantly different from the DMN-treated group.

478

479


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480 481

Table 2. Effects of CPS on DMN-induced body and liver weights in rats. Liver/Body weight (g/body weight) 4.21 ± 0.05

Body weight (g)

Liver weight (g)

Control

305 ± 28.4

12.6 ± 1.4

DMN 10mg/kg

198 ± 18.9

DMN + CPS 0.5 mg/kg

212 ± 20.6

6.6 ± 0.6

3.09 ± 0.45

DMN + CPS 1.0 mg/kg

251 ± 24.7 *

7.9 ± 0.7 *

3.52 ± 0.43 *

#

6.0 ± 0.5

#

2.93 ± 0.01

#

482 483 484

Results are expressed as the means ± SEM. # P < 0.05, significantly different from the control

485

group. * P < 0.05, significantly different from the DMN-treated group.

486 487 488



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489

Figure Legends

490

Figure 1. Schematic diagram of the experimental protocol. Rats were divided into four

491

groups of five rats each. Hepatic fibrosis was induced by dissolving DMN in sterile saline

492

(10 mg/kg body weight) and then injecting the rats intraperitoneally (i.p.) three times per

493

week for 4 weeks. Capsaicin (CPS) was dissolved in absolute ethanol, diluted in saline and

494

then administered to the rats intragastrically (i.g.) at doses of 0.5 and 1.0 mg/kg/day six times

495

per week for 4 weeks. Control and DMN-treated groups were administered saline alone (i.g.)

496

without CPS. The animals were euthanized on day 29.

497 498

Figure 2. Effects of CPS on DMN-induced histopathological changes and hepatic

499

collagen content. (A) Liver tissue was collected and fixed in 10% formaldehyde. Thin

500

sections (5 µm) were cut and then stained with hematoxylin and eosin (H&E) and Masson’s

501

trichrome (MT). α-SMA expression was detected immunohistochemically. (B) Fibrosis

502

scores were determined by morphometric analysis of the computerized images of MT-stained

503

liver sections. (C) Liver collagen content was determined by assaying total soluble collagen

504

using the Sircol collagen assay kit according to the manufacturer’s directions.

505

significantly different from the control group. * P < 0.05, significantly different from the

506

DMN-treated group.

#

P < 0.05,

507 508

Figure 3. Effects of CPS on DMN-induced α-SMA, collagen type I, MMP-2, and TIMP-

509

1 expression. (A) Total protein extracted from liver tissue was subjected to Western blotting

510

to determine α-SMA, collagen type I, and β-actin levels. (B) The intensities of the α-SMA

511

and collagen type I bands were measured using the NIH Image J program. (C) Total RNA

512

extracted from liver tissue was used in a RT-PCR to determine the levels MMP-2, TIMP-1,


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513

and β-actin mRNA. (D) The intensities of the MMP-2 and TIMP-1 RT-PCR bands were

514

measured using the NIH Image J program. # P < 0.05, significantly different from the control

515

group. * P < 0.05, significantly different from the DMN-treated group

516 517

Figure 4. Effects of CPS on DMN-induced TNF-α and NF-κB activation. (A) Total RNA

518

from liver tissue was subjected to RT-PCR to measure the levels of mRNAs encoding TNF-α

519

and β-actin. (B) Nuclear protein extracted from liver tissue was analyzed by Western blotting

520

to determine NF-κB and laminin B1 levels. Total protein extracted from liver tissue was also

521

subjected to Western blotting to determine IκBα and β-actin levels. (C) The intensities of the

522

bands were measured using the NIH Image J program. (D) Liver tissues were lysed and the

523

levels of p-NF-κB P65 (Ser536) and p-IκBα (Ser32) detected using PathScan Sandwich

524

ELISA kits.

525

significantly different from the DMN-treated group.

#

P < 0.05, significantly different from the control group. * P < 0.05,

526 527

Figure 5. Effects of CPS on the DMN-induced Smad2/3 phosphorylation and DMN-

528

induced reduction in Smad7 expression. (A) Total protein extracted from liver tissue was

529

subjected to Western blotting to determine Smad2 and Smad3 phosphorylation and Smad7

530

and β-actin levels. (B) The intensities of the bands were measured using the NIH Image J

531

program. # P < 0.05, significantly different from the control group. * P < 0.05, significantly

532

different from the DMN-treated group.

533 534

Figure 6. Effects of CPS on the DMN-induced increase in TGF-β1 expression and

535

reduction in PPARγ expression. (A) Total protein extracted from liver tissue was subjected

536

to Western blotting to determine TGF-β1, PPARγ, and β-actin levels. (B) The intensities of



Page 22 of 31 22

537

the bands were measured using the NIH Image J program. # P < 0.05, significantly different

538

from the control group. * P < 0.05, significantly different from the DMN-treated group.

539 540

Figure 7. Effects of CPS on TGF-β β 1-induced changes in α-SMA and collagen type I

541

expression via PPARγγ activation in HSC-T6 cells. (A) Cells were treated with various

542

concentrations of TGF-β1 for 24 h or with TGF-β1 (5 ng/mL) for 6, 12, and 24 h. (B–D)

543

Cells were pretreated with various levels of CPS for 1 h and then stimulated with TGF-β1 (5

544

ng/mL) for 24 h. Cells were pretreated with GW9662 (10 µM) for 1h and then with CPS (10

545

µM) for 1 h. Finally, cells were stimulated with TGF-β1 (5 ng/mL) for 24 h. Total protein

546

extracts were subjected to Western blotting to determine PPARγ, α-SMA, and β-actin levels.

547

Total RNAs from liver tissue were subjected to RT-PCR to measure the levels of mRNAs

548

encoding collagen type I and β-actin.

549 550

Figure 8. Effect of CPS on DMN-induced liver fibrosis. CPS reduced hepatotoxicity,

551

inflammation, and hepatic stellate cell (HSC) activation during hepatic fibrogenesis.

552

Especially, CPS ultimately inhibited HSC activation via a mechanism involving the TGF-

553

β/Smad2/3/7 and PPARγ signaling pathways. This is strong evidence that CPS may be useful

554

in the treatment of liver fibrosis. The arrows indicate stimulatory effects and the lines indicate

555

suppressive effects.

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DMN Capsaicin Oxidative stress 

PPAR Liver fibrosis  -SMA ↑ Col1 ↑ ECM ↑

Hepatotoxicity  ALT/AST ↑ Lipid peroxidation ↑

HSC activation Inflammation  NF-B ↑ TNF- ↑


Capsaicin Inhibits Dimethylnitrosamine-Induced Hepatic Fibrosis by

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