Curcumin modulates cannabinoid receptors in liver fibrosis in vivo and inhibits extracellular matrix expression in hepatic stellate cells by suppressing cannabinoid receptor type-1 in vitro

Curcumin modulates cannabinoid receptors in liver fibrosis in vivo and inhibits extracellular matrix expression in hepatic stellate cells by suppressing cannabinoid receptor type-1 in vitro

Zili Zhang a,b, Yao Guo a,b, She Zhang a,b, Yan Zhang a,b, Yuqing Wang a,b, Wenxia Ni a,b, Desong Kong a,b, Wenjing Chen d,n, Shizhong Zheng a,b,c,nn

a Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
b National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing 210023, China c Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Material Medical, Nanjing University of Chinese Medicine, Nanjing 210023, China
d Institute of Stomatology, Nanjing Medical University, Nanjing 210029, China

article info

Article history:

Received 31 May 2013
Received in revised form
11 September 2013
Accepted 19 September 2013 Available online 26 September 2013

Keywords:

Liver fibrosis
Hepatic stellate cell Curcumin Cannabinoid receptor Extracellular matrix

1. Introduction

Hepatic fibrosis is a serious healthcare problem with high morbidity and mortality. It is the result of wound-healing responses to repeated liver injury irrespective of etiology. With the develop- ment of the disease, excessive extracellular matrix (ECM) compo- nents are deposited in the liver, leading to portal hypertension, cirrhosis or hepatocellular carcinoma (Hernandez-Gea and Friedman, 2011). Numerous studies have established that following liver injury,

n Corresponding author at: Institute of Stomatology, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, China. Tel.: þ86 25 86798154;
fax:
þ86 25 86798188.

nn Corresponding author at: Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, China. Tel.: þ86 25 86798154; fax: þ86 25 86798188.

E-mail addresses: chenwenjing_orth@163.com (W. Chen), nytws@163.com (S. Zheng).

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.09.042

& 2013 Elsevier B.V. All rights reserved.

quiescent hepatic stellate cells (HSCs) undergo profound morpholo- gical and functional changes, and transdifferentiate into proliferative, contractile and chemotaxic myofibroblast-like cells, which function as the main ECM-producing cells contributing to the pathogenesis of liver fibrosis (Friedman, 2008). Reduction of ECM components expressed by HSCs is thus considered to be a primary therapeutic strategy for the treatment of hepatic fibrosis.

Continuing understanding of pathology of liver fibrosis has revealed that the endocannabinoid system plays a critical role in HSC pathobiology and pathogenesis of chronic liver injury (Tam et al., 2011). The endocannabinoid system comprises endocannabinoids such as arachidonoyl ethanolamide and 2-arachidonoyl- glycerol, and their corresponding receptors, namely cannabinoid receptors type 1 and 2 (CBR1 and CBR2). Compelling evidence indicates that CBR1 expression is confined to HSCs and vascular endothelium, whereas CBR2 is expressed by inflammatory cells in liver (Caraceni et al., 2008). Studies showed that CBR1 knockout mice were resistant to fibrogenesis induced by carbon

abstract

Activation of hepatic stellate cells (HSCs) is a pivotal event leading to extracellular matrix (ECM) overproduction during hepatic fibrogenesis. Compelling evidence indicates that cannabinoid receptors (CBRs) play an important role in chronic liver disease. Antagonism of hepatic CBR type 1 (CBR1) could be a novel therapeutic strategy for liver fibrosis. Our previous studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work was to examine the curcumin effect on CBRs system and its relevance to inhibition of ECM expression in HSCs. Our in vivo data demonstrated that curcumin ameliorated fibrotic injury, and downregulated CBR1 but upregulated CBR2 at both mRNA and protein levels in rat fibrotic liver caused by carbon tetrachloride. The subsequent in vitro investigations showed that curcumin reduced the mRNA and protein abundance of CBR1 in cultured HSCs and decreased the expression of three critical ECM proteins. Further analyses revealed that CBR1 agonist abrogated the curcumin inhibition of ECM expression, but CBR1 antagonist mimicked and reinforced the curcumin effects. Autodock simulations predicted that curcumin could bind to CBR1 with two hydrogen bonds. Collectively, our current studies revealed that curcumin reduction of liver fibrosis was associated with modulation of CBRs system and that antagonism of CBR1 contributed to curcumin inhibition of ECM expression in HSCs.

134 Z. Zhang et al. / European Journal of Pharmacology 721 (2013) 133140

tetrachloride (CCl4), thioacetamide or bile duct ligation (Teixeira- Clerc et al., 2006), whereas CBR2 knockout mice exhibited more progressive fibrosis after CCl4 treatment (Julien et al., 2005), suggesting that CBR1 and CBR2 mediate opposite effects in liver fibrogenesis. Clinically, patients with chronic hepatitis C and daily cannabis consumption displayed more severe fibrosis progression than non- or occasional consumers (Hezode et al., 2005, 2008; Ishida et al., 2008). These discoveries strongly suggest CBRs as new therapeutic targets for chronic liver diseases.

Currently, research identifying antifibrogenic agents that are innocuous is urgently needed. We previously documented that curcumin, the yellow pigment of turmeric in curry derived from the rhizome of the plant Curcuma longa, disrupted transforming growth factor-β (TGF-β) signaling and inhibited connective tissue growth factor expression, leading to inhibited HSC proliferation (Zheng and Chen, 2006, 2007). However, the underlying mechan- isms remain incompletely understood. Interestingly, a recent inves- tigation showed that curcumin exerted the antidepressant activity by targeting CBR1-mediated endocannabinoid signaling and brain nerve growth factor (Hassanzadeh and Hassanzadeh, 2012). Given the therapeutic potential of CBR1 antagonism for liver fibrosis (Teixeira-Clerc et al., 2006), we thus hypothesized that modulation of CBRs system could contribute to curcumin reduction of liver fibrosis and inhibition of ECM production in HSCs. We performed in vivo and in vitro experiments to test the hypothesis.

2. Materials and methods

2.1. Reagents and antibodies

Curcumin, N-arachidonoyldopamine (NADA), and AM251 were from Sigma (St Louis, MO, USA). All these compounds were dissolved in dimethylsulfoxide (DMSO; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) for experiments. Primary antibodies against α-SMA, α(I) procollagen, fibronectin were from Epitomics (San Francisco, CA, USA). Primary antibodies against CBR1, CBR2, and β-actin were from Bioworld Technology (Nanjing, China).

2.2. Experimental animal procedures

Male Sprague-Dawley rats (180220 g body weight) were obtained from Nanjing Medical University (Nanjing, China). A mixture of CCl4 (0.1ml/100gbodyweight) and olive oil [1:1 (w/v)] was used to induce liver fibrosis in rats. Thirty rats were randomly divided into five groups (six rats/group). Group 1 was the vehicle control in which rats were not administrated CCl4 or curcumin but intraperitoneally (i. p.) injected with olive oil. Group 2 was the CCl4 group in which rats were i.p. injected with CCl4 without curcumin treatment. Groups 35 were treatment groups in which rats were i.p. injected with CCl4 and orally given curcumin at 100, 200, and 400 mg/kg, respectively. Rats in groups 25 were i.p. injected with CCl4 every other day for 8 weeks. Curcumin was suspended in 1% sodium carboxyl methyl cellulose and given once daily by gavage during weeks 58. At the end of experiment, rats were sacrificed after being anesthetized by i.p. pentobarbital (50 mg/kg).

2.3. Liver histopathology

Liver histology and collagen deposition were examined using hematoxylineosin (HE) staining and masson staining, respec- tively, as we previously described (Fu et al., 2008). Representative views of liver sections were shown and quantified with Image J software.

2.4. Cell isolation and culture conditions

Primary HSCs were isolated from male Sprague-Dawley rats. Briefly, portal vein perfusion was begun after heparin administration. Phosphate buffered saline (PBS) at 37 1C was perfused at 10 ml/min to adequately blanch the liver. This was followed by 0.1% Pronase for 34 min and then 0.03% collagenase for 30 min with reperfusion at 5 ml/min of the latter from the inferior vena cava. The liver was subsequently removed, minced in 0.02% Pronase, and incubated in a shaking 37 1C water bath for 2030 min with deoxyribonuclease (10 μg/ml). The mixture was then centrifuged at 50g for 2 min to remove dead hepatocytes and undigested debris. The supernatant containing hepatic nonparenchymal cells was washed four times in PBS and then layered over a 25% preformed (30,000g for 15 min) Percoll gradient and centrifuged at 800g for 30 min. This gradient produced a top layer of yellowish white oily debris with a band of cells immediately beneath which contained HSCs. The HSC band was then washed two times and placed on a 45% unformed Percoll gradient and centrifuged at 15,000g for 20 min. This latter centrifu- gation removed potential bacterial contamination, leaving the cell band at the top of the tube. The cells were then washed two times in PBS and resuspended in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum (FBS; Sijiqing Biological Engineering Materials, Hangzhou, China). This culture medium was routinely used throughout primary and secondary cultures, and then changed into Dulbecco's modified eagle medium (DMEM; Invitrogen, Grand Island, NY, USA) with 10% FBS, 1% antibiotics, and grown in a 5% CO2 humidified atmosphere at 37 1C. HSCs at passages 48 were used in experiments.

2.5. Immunofluorescence staining

Staining of liver sections with primary antibodies against CBR1 and CBR2 was performed as we previously reported (Fu et al., 2008). For staining with cells, HSCs were seeded in 6-well plates and cultured in DMEM with 10% FBS for 24 h. HSCs were then treated with DMSO (0.02%, w/v) or curcumin at indicated con- centrations for 24 h. Staining was performed as we previously described (Zhang et al., 2013) and Hoechst 33342 reagent (Beyo- time Institute of Biotechnology, Haimen, China) was used to stain the nucleus. Representative micrographs are shown.

2.6. Real-time PCR

Total RNA was isolated from liver tissues or treated HSCs, and real-time PCR was performed as we previously described (Zhang et al., 2012). Glyceraldehyde phosphate dehydrogenase (GAPDH) was used as the invariant control. The following primer sequences were used in experiments: CBR1: (forward) 5-CTGATCCCACACC CTTTCAT-3, (reverse) 5-TTAAGGGCTCTGACGCTCAT-3; CBR2: (for- ward) 5-TTCCCCCTGATCCCCAACGACTA-3, (reverse) 5-CTCTCCAC TCCGCAGGGCATAAAT-3; GAPDH: (forward) 5-GGCCCCTCTGGAA AGCTGTG-3, (reverse) 5-CCGCCTGCTTCACCACCTTCT-3.

2.7. Western blot analyses

Total proteins were prepared from liver tissues or treated HSCs as we previously described (Zhang et al., 2013) with corresponding primary antibodies or β-actin as loading control. The levels of protein bands were densitometrically determined using Quantity One 4.4.1 (Bio-Rad Laboratories, Hercules, CA, USA). Representa- tive blots from three independent experiments are shown.

2.8. Molecular docking studies

Automated docking is widely used as an effective tool capable of quickly and accurately predicting biomolecular conformations and binding energies of proteinligand complexes in molecular design (Lybrand, 1995). The crystal structures of CBR1 (PDB code: 1LVQ) served as the docking template. Docking studies were conducted according to we previously described (Zhang et al., 2013). Briefly, crucial amino acids in the active sites of 1LVQ were identified, and essential hydrogen atoms were added and the water molecules were removed from the surface of the protein. The 3D structure of curcumin was constructed using Chem3D Ultra 8.0 software (Chemical Structure Drawing Standard; Cambridge Soft Corporation, USA) to obtain the standard 3D structure (pdb format). Then it was energetically minimized using MOPAC with 100 iterations and minimum RMS gradient of 0.10. A grid of 60 Å3, 0.375Å spacing was first computed in order to sample the binding sites. A flexible ligand docking was performed to this grid representation of the receptor binding site followed by scoring the receptorligand interaction. Docking studies were conducted using the AutoDock 4.2 (Cosconati et al., 2010). The software packages Accelrys Insight II were used to visualize and analyze the chemical structures. As a result of AutoDock calcula- tions, we obtained the output file with the proteinligand com- plexes with flexible residues and the ligands located within the binding pocket.

2.9. Statistical analyses

Data were presented as mean 7 S.D. and analyzed using SPSS16.0 software. Significance of difference was determined by one-way

receptors at protein level were consistent with that at mRNA level in fibrotic rats and rats receiving curcumin treatment (Fig. 2B and C). These data collectively showed that curcumin downregulated CBR1 and upregulated CBR2 in the fibrotic liver, possibly contributing to its antifibrotic properties.

3.3. Curcumin inhibits the expression of CBR1 and ECM proteins in cultured HSCs

Culturing freshly isolated HSCs is a well-established in vitro model for investigating antifibrotic intervention (Friedman, 2008). We sub- sequently used these cultured cells to investigate whether curcumin could inhibit CBR1 in HSCs, since studies have shown that the CBR1 is mainly expressed in HSCs and plays a pro-fibrogenic role in liver fibrosis (Tam et al., 2011). Cellular immunofluorescence staining showed that CBR1 was highly expressed in HSCs, but curcumin reduced its expression dose-dependently (Fig. 3A). Further examina- tions confirmed that curcumin could at both mRNA and protein levels reduced CBR1 expression in HSCs (Fig. 3B and C). We next found that curcumin reduced the expression of ECM components including α- SMA, α(1)procollagen, and fibronectin in HSCs (Fig. 3D). Taken together, these results demonstrated that curcumin inhibited the expression of CBR1 and ECM proteins in HSCs in culture.

3.4. Curcumin inhibits the expression of ECM proteins in HSCs possibly by targeting CBR1

To investigate the relationship between inhibition of CBR1 and reduction of ECM components in HSCs by curcumin, CBR1 selective agonist NADA and antagonist AM251 were used to perform gain- or loss-of-function analyses. The results demonstrated that ligand activation of CBR1 by NADA dose-dependently rescued the curcu- min downregulation of α-SMA, α(1)procollagen, and fibronectin (Fig. 4A). However, pharmacological inhibition of CBR1 by AM251 was found to reduce the expression of the three ECM proteins mimicking the curcumin effects; and combination of AM251 and curcumin resulted in more significant inhibitory effects (Fig. 4B). These data consistently indicated that CBR1 could be a molecular target, by suppressing which curcumin inhibited ECM expression in HSCs. It is known that the therapeutic activity of curcumin is attributed mainly to its unique chemical structure. We addi- tionally employed molecular docking studies to virtually predict the interactions between curcumin and CBR1. The surface mode of docking results showed that curcumin could be embedded into the binding pocket of CBR1, suggesting a favorable space compatibility between the two structures (Fig. 4C1). The ribbon mode of docking results showed that curcumin could interact with the amino acid residues located at the active site of CBR1 (Fig. 4C2). Further analyses demonstrated that two hydrogen bonds could be formed between the β-diketo oxygens and residues Asp 213 and Leu137, which might account for the potential affinity of curcumin for CBR1 (Fig. 4C3). These data suggested that curcumin could bind to CBR1 and confirmed that CBR1 could be a molecular target for curcumin inhibition of ECM expression in HSCs.

4. Discussion

Increasing studies have shown that the hepatic endocannabinoid system plays a critical role in HSC pathophysiology. The endocanna- binoid system consists of CBRs, endocannabinoids, and the enzymes involved in their biosynthesis and degradation. CBR1 is the most abundant receptors in the mammalian brain, but it is also expressed in peripheral tissues, including various cell types of the liver. The psychoactive properties of CBRs and the abundance of CBR1 in the

ANOVA with the post-hoc Dunnett's test. Values of P o 0.05 considered to be statistically significant.

3. Results

3.1. Curcumin attenuates CCl4-caused liver fibrosis in rats

were

We initially examined the ameliorative effects of curcumin on hepatic fibrotic injury in vivo. Curcumin treatment resulted in remark- able improvement in liver histology and collagen deposition evi- denced by ameliorated state of hepatic steatosis, necrosis, and fibrotic septa, and reduced positive-staining size in the liver of CCl4- treated rats (Fig. 1A). In addition, we detected the protein abundance of α-smooth muscle actin (α-SMA), α(1)procollagen, and fibronectin, three key ECM components during liver fibrosis (Fu et al., 2008). The results showed that the three markers were all significantly elevated in the fibrotic liver, but their expression was reduced dose-depen- dently by curcumin (Fig. 1B). These results consistently demonstrated that curcumin attenuated CCl4-caused liver fibrosis in vivo.

3.2. Curcumin modulates the expression of cannabinoid receptors in rat fibrotic liver

We next evaluated the effects of curcumin on the CBRs system (mainly the pro-fibrotic CBR1 and anti-fibrotic CBR2) in liver fibrosis. Real-time PCR analyses showed that CBR1 mRNA was significantly increased in the fibrotic liver, but treatment with curcumin reduced its mRNA expression in a dose-dependent manner. In contrast, hepatic CBR2 mRNA level was significantly decreased during fibro- genesis, but curcumin dose-dependently restored its gene expression (Fig. 2A). We additionally used Western blot and immunofluores- cence staining to determine the protein expression of CBR1 and CBR2 in rat liver. The results showed that the alterations of the two

Z. Zhang et al. / European Journal of Pharmacology 721 (2013) 133140 135

136 Z. Zhang et al. / European Journal of Pharmacology 721 (2013) 133140

Fig. 1. Curcumin attenuates CCl4-caused liver fibrosis in rats. Rats were grouped: group 1, vehicle control (no CCl4, no treatment); group 2, model group (with CCl4, no treatment); group 3, curcumin-treated group (100 mg/kgþCCl4); group 4, curcumin-treated group (200 mg/kgþCCl4); group 5, curcumin-treated group (400 mg/kgþCCl4). (A) Liver sections were stained with HE, and masson reagents for histological examination. Representative graphs are shown with quantification (n 1⁄4 6). Data are expressed as mean7S.D., ##Po0.01 vs group 1, nPo0.05 vs group 2, nnPo0.01 vs group 2. (B) Western blot analyses of fibrotic markers in liver tissues. β-Actin was used as an invariant control for equal loading. Representative blots are shown with densitometry (n1⁄46). Data are expressed as mean7S.D., #Po0.05 vs group 1, ##Po0.01 vs group 1, nPo0.05 vs group 2, nnPo0.01 vs group 2.

Fig. 2. Curcumin modulates the expression of cannabinoid receptors in rat liver with CCl4-caused fibrosis. Rats were grouped: group 1, vehicle control (no CCl4, no treatment); group 2, model group (with CCl4, no treatment); group 3, curcumin-treated group (100 mg/kgþCCl4); group 4, curcumin-treated group (200 mg/kgþCCl4); group 5, curcumin-treated group (400 mg/kgþCCl4). (A) Real-time PCR analyses of CBR1 and CBR2 in liver tissues. GAPDH was used as the invariant control for calculating fold changes in mRNA levels. Data are expressed as mean7S.D., ##Po0.01 vs group 1, nPo0.05 vs group 2, nnPo0.01 vs group 2, n1⁄46. (B) Western blot analyses of CBR1 and CBR2 in liver tissues. β-Actin was used as an invariant control for equal loading. Representative blots are shown with densitometry (n1⁄46). Data are expressed as mean7S.D., #Po0.05 vs group 1, ##Po0.01 vs group 1, nPo0.05 vs group 2, nnPo0.01 vs group 2. (C) Immunofluorescence analyses of CBR1 and CBR2 in liver tissues with quantification (n1⁄46). Data are expressed as mean7S.D., ##Po0.01 vs group 1, nPo0.05 vs group 2, nnPo0.01 vs group 2.

brain indicate that the endocannabinoid system is primarily a neuronal signaling system; therefore, evidence for the presence and functional importance of this system in the liver was unexpected (Caraceni et al., 2008; Tam et al., 2011). Our current results showed

that CBR1 was detectable in rat liver, which was consistent with several recent reports that documented low-level CBR1 expression in hepatocytes, HSCs and liver vascular endothelial cells (Jeong et al., 2008; Mukhopadhyay et al., 2010; Osei-Hyiaman et al., 2008).

Z. Zhang et al. / European Journal of Pharmacology 721 (2013) 133140 137

Fig. 3. Curcumin inhibits the expression of CBR1 and ECM proteins in HSCs. HSCs were treated with DMSO (0.02%, w/v) and curcumin at the indicated concentrations for 24 h. (A) Immunofluorescence analyses of CBR1. Hoechst reagent was used to stain the nucleus. Representative graphs are shown (n1⁄43). (B) Real-time PCR analyses of CBR1 transcripts. GAPDH was used as the invariant control for calculating fold changes in mRNA levels. Data are expressed as mean7S.D. (n1⁄43), nPo0.05 vs DMSO. (C) Western blot analyses of CBR1. β-Actin was used as an invariant control for equal loading. Representative blots are shown with densitometry (n1⁄43). Data are expressed as mean7S.D., nPo0.05 vs DMSO, nnPo0.01 vs DMSO. (D) Western blot analyses of fibrotic markers. β-Actin was used as an invariant control for equal loading. Representative blots are shown with densitometry (n1⁄43). Data are expressed as mean7S.D., nPo0.05 vs DMSO, nnPo0.01 vs DMSO.

However, CBR1 expression was significantly increased in the fibrotic liver, which was in agreement with the clinical observation that CBR1 was present in human hepatocytes and in the whole human liver, with increased expression noted in patients with hepatocellular carcinoma or primary biliary cirrhosis (Floreani et al., 2010; Xu et al., 2006). On the other hand, CBR2 is expressed primarily in immune and hematopoietic cells and has also been detected in the liver in certain pathological states (Ashton et al., 2007; Julien et al., 2005). We herein found that CBR2 expression was considerably repressed in the fibrotic liver, suggesting that the loss of hepatic CBR2 could be associated with liver fibrosis. This could be confirmed

by the finding that mice lacking CBR2 developed significantly enhanced liver fibrosis compared with the wide type mice (Julien et al., 2005). During liver fibrogenesis, endogenous activation of CBR2 could limit the progression of fibrosis by reducing accumulation of liver fibrogenic cells. Overall, our present results together with others strongly indicated that selective modulation of CBRs may open a new approach for the treatment of liver fibrosis.

We have previously documented that curcumin could interrupt some pro-fibrogenic pathways in HSCs (Zheng and Chen, 2006, 2007). However, the underlying mechanisms are not fully under- stood. In this report, we demonstrated that curcumin protected

138 Z. Zhang et al. / European Journal of Pharmacology 721 (2013) 133140

Fig. 4. Curcumin inhibits ECM expression in HSCs by suppressing CBR1. (A) HSCs were treated with DMSO (0.02%, w/v), curcumin, NADA at the indicated concentrations for 24 h. Western blot analyses of fibrotic markers. β-Actin was used as an invariant control for equal loading. Representative blots are shown with densitometry (n 1⁄4 3). Data are expressed as mean7S.D., #Po0.05 vs DMSO, ##Po0.01 vs DMSO, nPo0.05 vs curcumin alone. (B) HSCs were treated with DMSO (0.02%, w/v), curcumin, AM251 at the indicated concentrations for 24 h. Western blot analyses of fibrotic markers. β-Actin was used as an invariant control for equal loading. Representative blots are shown with densitometry (n1⁄43). Data are expressed as mean7S.D., nPo0.05 vs DMSO, nnPo0.01 vs DMSO. (C) Docking curcumin to the X-ray structure of CBR1. (1) Surface mode of docking results showing the embedment of curcumin into the binding pocket of CBR1. (2) Ribbon mode of docking results showing a stereo-view of curcumin binding to the active site of CBR1. (3) Interactions of curcumin with the amino acid residues located at the active site of CBR1. Hydrogen bonds are indicated by white arrows.

the rat liver from CCl4-caused fibrosis, which was in agreement with our prior data that curcumin reduced the amount of HSCs in vivo (Fu et al., 2008). More importantly, curcumin was clearly demonstrated to modulate the CBRs system in the fibrotic liver. Curcumin dose-dependently downregulated the abundance of CBR1, which is pro-fibrogenic, and restored the expression of CBR2, which is anti-fibrogenic in the fibrotic liver. These effects might contribute to the antifibrotic properties of curcumin and indicated that pharmacological modulation of CBRs system for management of liver fibrosis could be feasible. It was indeed reported that pharma- cological inhibition of CBR1 with rimonabant, a CBR1 antagonist, reduced the expression of TGF-β and α-SMA, and improved liver histology in mice (Siegmund and Schwabe, 2008; Wasmuth and Trautwein, 2007). Moreover, a recent study showed that CBR2 agonist JWH-015 alleviated portal hypertension, severity of portosystemic collaterals and mesenteric angiogenesis, intrahepatic angiogenesis, and fibrosis in rats with common bile duct ligation-induced liver fibrosis (Huang et al., 2012). Another study with CBR2 selective agonist JWH-133 demonstrated similar results in cirrhotic rats (Munoz-Luque et al., 2008). Our present data revealed that hepatic CBRs system could be regulated by curcumin, which could be a novel

mechanism for curcumin treatment of liver fibrosis. However, it is known that hepatic fibrosis is the result of reiterated liver injury due to various etiologies including parasitic disease, chronic infec- tion by viral agents (mainly hepatitis B and C viruses), or metabolic, toxic/drug-induced and autoimmune causes (Parola et al., 2008). Moreover, the endocannabinoid system is also involved in immu- noregulation. For example, administration of endocannabinoids or use of inhibitors of enzymes that break down the endocannabinoids led to immunosuppression and recovery from immune-mediated injury to organs such as the liver (Nagarkatti et al., 2009). Targeting cannabinoid receptors using exogenous or endogenous cannabi- noids might constitute novel therapeutic modalities to treat immune-mediated liver inflammation (Hegde et al., 2008). There- fore, manipulation of hepatic endocannabinoid system implied that curcumin might be more useful for the management of autoimmune-related liver fibrosis.

At molecular level, a large number of signaling pathways have been shown to contribute to the pro-fibrogenic properties of HSCs and the subsequent accumulation of ECM components in the liver during fibrogenesis (Friedman, 2008). Emerging data suggest that the CBRs system especially the CBR1 is an important part of this

complex signaling network during liver diseases (Teixeira-Clerc et al., 2006). Recent studies showed that CBR1 enhanced portal hypertension by promoting splanchnic vasodilation and increase steatogenesis associated with obesity (Batkai et al., 2001; Osei-Hyiaman et al., 2005; Ros et al., 2002). Pharmacological antagonism of CBR1 could be a new strategy for the treatment of liver fibrosis. We herein performed mechanistic analyses to examine the curcumin effect on CBR1 in HSCs and its role in reduction of ECM expression in HSCs. The results showed that curcumin could reduce the expression of CBR1 at both gene and protein levels in HSCs. Curcumin reduction of ECM production could be abolished by pharmacological activation of CBR1 but enhanced by pharmacological inhibition of CBR1. These data suggested that suppressing CBR1 could be required for curcumin to inhibit ECM gene expression in HSCs, and reinforced the role of CBR1 as a target molecule for reducing ECM expression. The availability of 3D protein structures enables the molecular docking strategy to be increasingly considered to obtain insights into the interplay between protein and ligand (Lybrand, 1995). We here employed this approach to predict the potential ligand-binding mode of curcumin to CBR1 using AutoDock 4.2, which uses an automated and robust docking algorithm based on the Lamarckian genetic algorithm and is a power- ful tool for calculating the position of docked ligand and flexible residues moved in the process of interaction and for comparing the energies of the interaction in different conformations and determining the best fit (Cosconati et al., 2010). The AutoDock scores are ranked taking into account the ligandreceptor interaction energy, conforma- tional strain energy of the ligand, conformational entropy loss and desolvation effects. Our current docking results revealed that curcu- min could interact with the amino acid residues of CBR1 with two hydrogen bonds. The docking results indicated that curcumin could be a ligand for CBR1. This could be explained by the fact that curcumin as a polyphenolic natural product structurally contains three important functionalities: an aromatic o-methoxy phenolic group, α,β-unsatu- rated β-diketo moiety and a flexible seven-carbon linker. These moieties make curcumin interact with a number of biomolecules through covalent and non-covalent binding including hydrogen bond- ing; the flexibility of the linker group is also responsible for the optimal confirmations in interaction with proteins (Priyadarsini, 2013). Our data herein preliminarily identified CBR1 as a target molecule of curcumin, and were also consistent with the experimental data that curcumin suppressed CBR1 leading to reduced ECM production in HSCs. Given that curcumin exhibits therapeutic activity against a number of diseases, we did not exclude the possibility that curcumin could bind to other biomolecules to exert its antifibrotic properties. Despite so, this docking study provided valuable insight into the molecular basis of curcumin, and it is of interest to seek more solid evidence to validate CBR1 as the direct molecular target for curcumin.

In summary, our current study demonstrated that curcumin attenuated fibrotic injury and modulated the CBRs system in rat fibrotic liver. The in vitro data showed that curcumin inhibited CBR1 resulting in reduced ECM expression in HSCs. Molecular docking investigations indicated that the CBR1 could be a target molecule for curcumin effects. Our results provided novel insights into curcumin inhibition of ECM expression in HSCs implicated in antifibrotic therapy.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (81270514, 30873424), the Doctoral Discipline Foundation of the Ministry of Education of China (20103 237110010), Jiangsu Natural Science Foundation (BK2008456), the Project for Jiangsu Basic Research Natural Science Foundation (BK2012528), the Project for Jiangsu Basic Research Natural Science

Foundation (BK2012528), the Project for Supporting Jiangsu Provincial Talents in Six Fields (2009-B-010), the Open Program of Jiangsu Key Laboratory of Integrated Acupuncture and Drugs (KJA200801), the Open Project Program of the National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Med- icine (2011ZYX4-008), the Eleven-FiveNational Science and Tech- nology Supporting Program (2008BAI51B02), and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (2011-137).

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