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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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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:

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ABSTRACT

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

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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

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

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

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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 ±

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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

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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

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TGF-β1/Smad pathway via PPARγ activation.

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

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Introduction

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

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endogenous metabolites and toxicants.

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

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

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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

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

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accumulation, and drug abuse.

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

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remodeling. Activated HSCs increase the proliferation of phenotypically transformed

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

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

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

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expression of genes involved in ECM synthesis and deposition.

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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

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

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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)

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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

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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

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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-

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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

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

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Korea).

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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

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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

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

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

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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

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

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Biochemical analysis and ELISA

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

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

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

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

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

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

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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

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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

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Hepatic collagen content

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

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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%

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

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Semi-quantitative RT-PCR

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

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

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as described previously.

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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

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Hepatic protein expression levels were determined by Western blotting.

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were obtained using NIH Image J software.

14

Band densities

149 150

Statistical analysis

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

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

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than 0.05 indicates statistical significance.

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RESULTS

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CPS inhibited hepatotoxicity in DMN-treated rats.

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

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

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fibrosis

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

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

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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).

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

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

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

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

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

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

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

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transactivation of HSCs to myofibroblasts. CPS treatment dramatically improved the

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

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

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

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

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

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

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

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

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

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

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

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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

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pathogenesis of chronic liver diseases.

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

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

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

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

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

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

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

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to translocate to the nucleus.

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

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

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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

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

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

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

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Smad activation, and collagen expression.

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

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

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

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

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Smad7 inactivation.

21

CPS treatment dose-dependently inhibited the

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Effects of CPS on PPARγγ expression

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

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

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

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

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

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

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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

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

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

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with liver fibrosis.

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DISCUSSION

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

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

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

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

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factors, together with enhanced ECM deposition.

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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

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

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

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

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

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

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

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

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fibrosis as the experimental animal model.

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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,

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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

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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

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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

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and the

hepatic lipid

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effect of CPS in the setting of chronic liver diseases.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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reducing hepatic inflammation and therefore liver fibrosis.

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

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

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Together, these

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

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

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

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the TGF-β-responsive element.

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

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previously reported in an experimental animal model of liver fibrosis.

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

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

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

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with CPS (Figure 8).

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

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

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fibrosis.

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

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

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

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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.

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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

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Our

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gastrointestinal stress,

308

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

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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

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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.

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References (1) Ingawale, D. K.; Mandlik, S. K.; Naik, S. R., Models of hepatotoxicity and the underlying cellular, biochemical and immunological mechanism(s): a critical discussion. Environmental toxicology and pharmacology 2014, 37, 118-33. (2) Shin, S. M.; Yang, J. H.; Ki, S. H., Role of the Nrf2-ARE pathway in liver diseases. Oxidative medicine and cellular longevity 2013, 2013, 763257. (3) Duarte, S.; Baber, J.; Fujii, T.; Coito, A. J., Matrix metalloproteinases in liver injury, repair and fibrosis. Matrix biology : journal of the International Society for Matrix Biology 2015, 44-46, 147-56. (4) Karsdal, M. A.; Manon-Jensen, T.; Genovese, F.; Kristensen, J. H.; Nielsen, M. J.; Sand, J. M.; Hansen, N. U.; Bay-Jensen, A. C.; Bager, C. L.; Krag, A.; Blanchard, A.; Krarup, H.; Leeming, D. J.; Schuppan, D., Novel insights into the function and dynamics of extracellular matrix in liver fibrosis. American journal of physiology. Gastrointestinal and liver physiology 2015, 308, G807-30. (5) Pinzani, M., Pathophysiology of Liver Fibrosis. Digestive diseases 2015, 33, 492-7. (6) Ding, N.; Yu, R. T.; Subramaniam, N.; Sherman, M. H.; Wilson, C.; Rao, R.; Leblanc, M.; Coulter, S.; He, M.; Scott, C.; Lau, S. L.; Atkins, A. R.; Barish, G. D.; Gunton, J. E.; Liddle, C.; Downes, M.; Evans, R. M., A vitamin D receptor/SMAD genomic circuit gates hepatic fibrotic response. Cell 2013, 153, 601-13. (7) Hong, S. W.; Jung, K. H.; Lee, H. S.; Zheng, H. M.; Choi, M. J.; Lee, C.; Hong, S. S., Suppression by fucoidan of liver fibrogenesis via the TGF-beta/Smad pathway in protecting against oxidative stress. Bioscience, biotechnology, and biochemistry 2011, 75, 833-40. (8) Lin, X.; Chen, Y.; Lv, S.; Tan, S.; Zhang, S.; Huang, R.; Zhuo, L.; Liang, S.; Lu, Z.; Huang, Q., Gypsophila elegans isoorientin attenuates CCl(4)-induced hepatic fibrosis in rats via modulation of NF-kappaB and TGF-beta1/Smad signaling pathways. International immunopharmacology 2015, 28, 305-12. (9) Wang, C. Y.; Liu, Q.; Huang, Q. X.; Liu, J. T.; He, Y. H.; Lu, J. J.; Bai, X. Y., Activation of PPARgamma is required for hydroxysafflor yellow A of Carthamus tinctorius to attenuate hepatic fibrosis induced by oxidative stress. Phytomedicine : international journal of phytotherapy and phytopharmacology 2013, 20, 592-9. (10)Zhang, X.; Han, X.; Yin, L.; Xu, L.; Qi, Y.; Xu, Y.; Sun, H.; Lin, Y.; Liu, K.; Peng, J., Potent effects of dioscin against liver fibrosis. Scientific reports 2015, 5, 9713. (11)Greiner, A. K.; Papineni, R. V.; Umar, S., Chemoprevention in gastrointestinal physiology and disease. Natural products and microbiome. American journal of physiology. Gastrointestinal and liver physiology 2014, 307, G1-15. (12)Zhang, A.; Sun, H.; Wang, X., Recent advances in natural products from plants for treatment of liver diseases. European journal of medicinal chemistry 2013, 63, 570-7. (13)Lee, M. F.; Liu, M. L.; Cheng, A. C.; Tsai, M. L.; Ho, C. T.; Liou, W. S.; Pan, M. H., Pterostilbene inhibits dimethylnitrosamine-induced liver fibrosis in rats. Food chemistry 2013, 138, 802-7. (14)Choi, J. H.; Jin, S. W.; Kim, H. G.; Khanal, T.; Hwang, Y. P.; Lee, K. J.; Choi, C. Y.; Chung, Y. C.; Lee, Y. C.; Jeong, H. G., Platycodi Radix attenuates dimethylnitrosamine-induced liver fibrosis in rats by inducing Nrf2-mediated antioxidant enzymes. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association 2013, 56, 231-9. (15)Moragas, A.; Garcia-Bonafe, M.; Sans, M.; Toran, N.; Huguet, P.; Martin-Plata, C.,

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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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nutrition et metabolisme 2011, 36, 201-9. (30)Zhao, Q.; Qin, C. Y.; Zhao, Z. H.; Fan, Y. C.; Wang, K., Epigenetic modifications in hepatic stellate cells contribute to liver fibrosis. The Tohoku journal of experimental medicine 2013, 229, 35-43. (31)Han, S. S.; Keum, Y. S.; Chun, K. S.; Surh, Y. J., Suppression of phorbol esterinduced NF-kappaB activation by capsaicin in cultured human promyelocytic leukemia cells. Archives of pharmacal research 2002, 25, 475-9. (32)Lee, I. O.; Lee, K. H.; Pyo, J. H.; Kim, J. H.; Choi, Y. J.; Lee, Y. C., Antiinflammatory effect of capsaicin in Helicobacter pylori-infected gastric epithelial cells. Helicobacter 2007, 12, 510-7. (33)Joung, E. J.; Li, M. H.; Lee, H. G.; Somparn, N.; Jung, Y. S.; Na, H. K.; Kim, S. H.; Cha, Y. N.; Surh, Y. J., Capsaicin induces heme oxygenase-1 expression in HepG2 cells via activation of PI3K-Nrf2 signaling: NAD(P)H:quinone oxidoreductase as a potential target. Antioxidants & redox signaling 2007, 9, 2087-98. (34)Hung, W. L.; Tsai, M. L.; Sun, P. P.; Tsai, C. Y.; Yang, C. C.; Ho, C. T.; Cheng, A. C.; Pan, M. H., Protective effects of garcinol on dimethylnitrosamine-induced liver fibrosis in rats. Food & function 2014, 5, 2883-91. (35)Yao, Q. Y.; Xu, B. L.; Wang, J. Y.; Liu, H. C.; Zhang, S. C.; Tu, C. T., Inhibition by curcumin of multiple sites of the transforming growth factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by carbon tetrachloride in rats. BMC complementary and alternative medicine 2012, 12, 156. (36)Sahebkar, A.; Chew, G. T.; Watts, G. F., New peroxisome proliferator-activated receptor agonists: potential treatments for atherogenic dyslipidemia and nonalcoholic fatty liver disease. Expert opinion on pharmacotherapy 2014, 15, 493-503. (37)Hazra, S.; Xiong, S.; Wang, J.; Rippe, R. A.; Krishna, V.; Chatterjee, K.; Tsukamoto, H., Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells. The Journal of biological chemistry 2004, 279, 11392-401. (38)Miyahara, T.; Schrum, L.; Rippe, R.; Xiong, S.; Yee, H. F., Jr.; Motomura, K.; Anania, F. A.; Willson, T. M.; Tsukamoto, H., Peroxisome proliferator-activated receptors and hepatic stellate cell activation. The Journal of biological chemistry 2000, 275, 3571522. (39)Fu, Y.; Zheng, S.; Lin, J.; Ryerse, J.; Chen, A., Curcumin protects the rat liver from CCl4-caused injury and fibrogenesis by attenuating oxidative stress and suppressing inflammation. Molecular pharmacology 2008, 73, 399-409. (40)Zheng, S.; Chen, A., Curcumin suppresses the expression of extracellular matrix genes in activated hepatic stellate cells by inhibiting gene expression of connective tissue growth factor. American journal of physiology. Gastrointestinal and liver physiology 2006, 290, G883-93. (41)Bitencourt, S.; de Mesquita, F. C.; Caberlon, E.; da Silva, G. V.; Basso, B. S.; Ferreira, G. A.; de Oliveira, J. R., Capsaicin induces de-differentiation of activated hepatic stellate cell. Biochemistry and cell biology = Biochimie et biologie cellulaire 2012, 90, 683-90. (42)van Avesaat, M.; Troost, F. J.; Westerterp-Plantenga, M. S.; Helyes, Z.; Le Roux, C. W.; Dekker, J.; Masclee, A. A.; Keszthelyi, D. Capsaicin-induced satiety is associated with gastrointestinal distress but not with the release of satiety hormones. The American journal of clinical nutrition 2016, 103, 305-13

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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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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.

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Figure Legends

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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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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

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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- ↑

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