Abstract. Xiaoyu Wei Yatang Chen 2 * Wenxiang Huang 2 *

Transcription

1 Research Communication Ginsenoside Rg1 ameliorates liver fibrosis via suppressing epithelial to mesenchymal transition and reactive oxygen species production in vitro and in vivo Xiaoyu Wei Yatang Chen 2 * Wenxiang Huang 2 * 1 1 Department of Infectious Diseases, Yongchuan Hospital of Chongqing Medical University, Chongqing, People s Republic of China 2 Department of Infectious Diseases, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People s Republic of China Abstract Liver fibrosis remains a major cause of morbidity and mortality with a complicated etiology and notorious complications, but there is a lack of efficacious therapeutics. Epithelial to mesenchymal transition (EMT) has a central role in the course of liver fibrosis, and thus, prevention of the development of EMT may control and even reverse liver fibrosis. This study aimed to examine the beneficial effect of ginsenoside Rg1, a phrenological active component isolated from Ginseng, and interrogate the mechanism in vitro and in vivo, with a focus on transforming growth factor-b (TGF-b)/Smad and Nrf2- mediated signaling pathways. We employed a TGF-b-induced EMT model in HSC-T6 cells and CCl 4 -induced liver fibrosis in animal. We found that ginsenoside Rg1 significantly reduced cell proliferation and reversed transforming growth factor-b (TGF-b)-induced EMT in HSC-T6 cells; and ginsenoside Rg1 induced cell apoptosis, Ginsenoside Rg1 decreased the cellular level of collagen I and III in HSC-T6 cells, indicating the amelioration of fibrosis. We showed that ginsenoside Rg1 reduced cellular reactive oxygen species (ROS). We also observed similar beneficial effects of ginsenoside Rg1 in vivo. The data showed that ginsenoside Rg1 decreased the level of alanine aminotransferase and aspartate aminotransferase, and collage type IV (IV-C), hyaluronic acid, and laminin in carbon tetrachloride (CCl4)-induced liver fibrotic model. Mechanistically, we showed that ginsenoside Rg1 tuned down the TGF-b/Smad and stimulated Nrf-2 nuclear translocation, which could explain the beneficial effects. In aggregate, our results demonstrate that ginsenoside Rg1 exhibits a protective effect on liver fibrosis via suppressing EMT and cellular ROS level. VC 2018 BioFactors, 00(00): , 2018 Keywords: ginsenoside Rg1; liver fibrosis; epithelial to mesenchymal transition; TGF-b/Smad signal Abbreviations: EMT, epithelial to mesenchymal transition; TGF-b, transforming growth factor-b; Nrf2, nuclear factor E2-related factor 2; ALT, alanine aminotransferase; AST, aspartate aminotransferase; IV-C, collage type IV; HA, hyaluronic acid; LN, laminin; CCl4, carbon tetrachloride; ROS, reactive oxygen species; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein. VC 2018 International Union of Biochemistry and Molecular Biology Volume 00, Number 00, Month/Month 2018, Pages *Address for correspondence: Prof. Wenxiang Huang, MD, and Prof. Yatang Chen, MD, Department of Infectious Diseases, The First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road,Yuzhong, Chongqing , People s Republic of China. s: wenxiang_huang@hotmail.com, cyt6535@ vip.sina.com. Wenxiang Huang and Yatang Chen contributed equally to this study. Received 3 February 2018; accepted 5 April 2018 DOI /biof.1432 Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com) BioFactors 1

2 BioFactors 1. Introduction Liver fibrosis is a major cause of morbidity and mortality worldwide, with many clinical notorious complications, such as portal hypertension, encephalopathy, and liver failure [1 3]. Notably, liver fibrosis substantially accelerates the risk of hepatocellular carcinoma, placing a substantial burden onto individual, society, and healthcare system [1,3]. The etiology of liver fibrosis is complicated with many contributing factors [3,4]. For example, viral infection is the most common cause. Hepatitis B virus is the major type of hepatitis virus with a substantial contribution to liver fibrosis in China [5,6]. In the United States, viral infection affects about 1 2% of the US population [7]; and the resultant cirrhosis is projected to reach 45% of those infected with hepatitis C virus patients in 2030 [8]. Also, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis contribute to liver fibrosis [9]. In addition, alcohol-induced disease, drug-induced toxicity, other liver infections (e.g., schistosomiasis), immune-mediated liver diseases (e.g., autoimmune hepatitis), metabolic disorders (e.g., lipid, glycogen, or metal storage disorders), and cholestasis (e.g., secondary biliary cirrhosis) all accelerate liver fibrosis. Thus, there is an urgent need to develop therapeutics for liver fibrosis treatment with well-elucidated molecular mechanism. To date, extensive efforts have been devoted to deciphering the underlying mechanism for the onset, development, and progression of liver fibrosis which is the consequence of a sustained wound-healing response to sustained risk factors [3,10 12], in which epithelial to mesenchymal transition (EMT) process is the dominant event and may have a central role in liver fibrosis [13]. A wealth of evidence shows that EMT plays a pivotal role in a number of pathophysiological conditions including fibrosis [14]. Transforming growth factor-b (TGF-b) and its related signaling pathway is a major driver of EMT [15], and many of the upstream regulatory factors and downstreamresponsive effectors of EMT have been identified and a large body of evidence suggests that targeting EMT is a promising strategy for fibrotic disease management [14]. Therefore, prevention of the development of EMT would be a promising therapeutic strategy to control and even reverse liver fibrosis. Ginseng is a traditional Chinese medicine and the root has been used for more than 2,000 years with numerous beneficial actions [16]. For example, the pharmacological effects of ginseng have been well demonstrated in the central nervous, cardiovascular, endocrine, and immune systems. Also, it has antineoplastic, antistress, and antioxidant activity [16,17]. These beneficial effects can be attributed to its multiple pharmacologically active components, and the most pharmacological actions of ginseng are attributed to ginsenosides, including ginsenoside Rg1 (Fig. 1A). Thus, this study mainly focused on ginsenoside Rg1, a compound isolated from traditional Chinese FIG 1 Ginsenoside Rg1 decreases cell viability. (A) Chemical structural of ginsenoside Rg-1. (B) Effect of TGF-b on HSC-T6 cell viability. Cells were treated with TGF-b at concentrations of 2, 4, 6, 8, 10, and 12 ng/ml for 48 h. The cell viability was measured using CCK-8. (B) Effect of ginsenoside Rg1 on HSC-T6 cell viability. Cells were treated with ginsenoside Rg-1 at concentrations of 20, 40, 60, 80, and 160 lg/ml in the presence or absence of TGF-b for 48 h. The cell viability was measured using CCK-8. (D) Effect of ginsenoside Rg1 and TGF-b on HSC-T6 cell growth. Images were taken under light microscope after 48 h of exposure of ginsenoside Rg1 (40 lg/ml) and TGF-b (1 ng/ml). Magnification: 2003, **P < The data were retrieved from three independent assays and analyzed by one-way analysis of variance (ANOVA) followed by Tukey s multiple comparison. 2 Ginsenoside Rg1 ameliorates liver fibrosis

3 herbal medicine Panax ginseng Meyer, to study the hepatoprotective effects and underlying mechanisms of ginsenoside Rg1 in vitro and in vivo. 2. Materials and methods 2.1. Chemicals and reagents Ginsenoside Rg1, TGF-b, Cell Counting Kit-8 (CCK-8), Dulbecco s phosphate buffered saline (D-PBS), protease inhibitor and phosphatase inhibitor cocktails, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco s modified Eagle medium (DMEM) was purchased from Corning Cellgro (Herndon, VA). 6-Diamidino-2-phenylindole (DAPI) was bought from Invitrogen (Carlsbad, CA). The annexin V:phycoerythrin (PE) apoptosis was purchased from BD Biosciences (San Jose, CA). The polyvinylidene difluoride (PVDF) membrane was bought from Millipore (Bedford, MA). Western blotting substrate and Pierce BCA protein assay kit were bought from Thermo Scientific (Hudson, NH). Primary antibodies against Nrf2, Lamin B, collagen I, collagen III, E- cadherin, vimentin, TGF-b, B-cell lymphoma 2 (Bcl-2), and Bcl-2-like protein4/bcl-2-associated X protein (Bax) were all purchased from Cell Signaling Technology (Beverly, MA). The antibody against b-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) Cell line and cell culture The HSC-T6 rat hepatic stellate cell line was obtained from Sigma-Aldrich and cells were maintained in DMEM supplemented with heat-inactivated 10% FBS and 1% penicillin/streptomycin in a 5% CO 2 /95% air-humidified incubator at 378C. Ginsenoside Rg1 was dissolved in diwater and freshly diluted to the predetermined concentrations with culture medium. - Transforming growth factor-b was dissolved by following the manufacturer s introduction. HSC-T6 cells were treated with ginsenoside Rg1 or TGF-b as indicated Cell proliferation assay The effect of ginsenoside Rg1 on cell viability was examined using CCK-8. Briefly, HSC-T6 cells were seeded in 96-well culture plates at a density of 8 3 l0 3 /well in 100 ll medium. After cells were attached, the cells were treated with ginsenoside Rg1 and TGF-b alone or in combination at indicated concentrations. The control cells received the vehicle only. After 24- or 48-h incubation, 10 ll of CCK-8 reagent was added to each well and cultured for another 4 h. Then, the absorbance was measured at a wavelength of 450 nm using a Synergy H4 Hybrid microplate reader (BioTek, Winooski, VT). The IC 50 values were determined using the relative viability over Rg1 concentration curve Measurement of intracellular ROS Intracellular reactive oxygen species (ROS) were monitored using the fluorescent probe CM-H2DCFDA, which was dissolved in DMSO as a concentrate and diluted to 0.1% of DMSO. In the presence of ROS, intracellular esterases cleave acetate groups from CM-H2DCFDA, and CMH 2DCFDA was oxidized to highly fluorescent dichlorofluorescein (DCF) in cells. After one wash with PBS, HSC-T6 were incubated with 25 lm CM-H 2DCFDA at 378C for 30 min, after that, the cells were treated with indicated concentrations of H 2 O 2, TGF-b, or ginsenoside Rg1. Cellular fluorescence intensity was quantified using a plate reader with excitation at 485 nm and emission at 530 nm Animal study Twenty male Kunming mice (male, g, 4 6 weeks) were used to establish the model of liver fibrosis and purchased from animal center of Chongqing Medical University (Chongqing, China). Kunming mice, a type of mouse commonly used in laboratory testing, was first originated from Swiss mice. It is commonly used for toxicology and neurology studies owing to its high yield, good quality, resistance, and strong adaptability. Particularly, it is the most highly produced in China nowadays for laboratory research in pharmacology and toxicology studies [18,19]. All animals were housed in plastic cages containing wood shaving and maintained in a room at C with a 12-h light cycle with free access to standard laboratory diet and water. The animal study protocol was approved by the Ethics Committee for Animal Experiments of Chongqing Medical University, Chongqing, China. The mice were randomly divided into four groups (n 5 5): control group, model group, ginsenoside Rg1 group (30 and 60 mg/kg), and treatment group. The animals weight were monitored prior to the beginning of experiment and then checked two times per week (Monday and Thursday) during the period of experiment. Animals were subcutaneous injected 10% CCl 4 in a 1:9 mixture with olive oil (0.4 ml/kg) alone, or with control solution, ginsenoside Rg1 (30 mg/kg), and ginsenoside Rg1 (mg/kg), twice a week for 6 weeks as described previously [20]. The MRI was performed in the course of treatment. At the end of experimental period, all animals were anesthetized by injecting 3% chloral hydrate 1 day after the last dose of administration. If the weight loses 10%, animals will be euthanized. The animal will be immediately euthanatized if other symptoms such as pale mucous membranes, shivering, failure to respond to stimuli, piloerection, matted hair coat, soiled anogenital/vent area, and vocalization are observed. Liver tissue and blood samples were collected and all the samples were stored at 2808C freezer for further analysis Histopathologic study The liver samples were collected for routine histopathologic examination. After flushing with cold PBS, the tissue was fixed in 4% paraformaldehyde for 72 h at 48C. In total, 4 lm tissue sections were subjected to H&E and Masson staining, and followed by microscopic examination (Nikon Eclipse 50i; Kanagawa, Japan). Wei et al. 3

4 BioFactors 2.7. Immunoassays Cytoplasm and nuclear proteins were extracted from cells using Cytoplasm and Nuclear Protein Extraction Kit according to the manufacturer s instruction (Thermo). Immunohistochemistry and immunocytochemistry were performed to examine the expression of TGF-b, collagen I, and E-cadherin in cells according to the manufacturer s instructions. Western blotting assays were also performed using whole-cell lysates. Cell lysates were prepared and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred onto PVDF membrane, and membranes probed primary antibodies including Nrf2, Lamin B, Bcl-2, Bax, collagen I, collagen III, TGF-b, Smad2/3, p-smad2/3, E-cadherin, vimentin, and actin overnight at 48C, then blotted with corresponding secondary antibodies. Visualization was accomplished using BioRad ChemiDocTM XRS system (Hercules, CA) with electrochemiluminescence substrate. Protein levels were normalized to match densitometric values of internal controls. In addition, ELISA was performed to examine the level of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (liver function parameters) and IV-C, hyaluronic acid (HA), and laminin (LN) (liver fibrosis parameters) according to the manufacturer s instructions Statistical analysis Data are expressed as the mean 6 standard deviation (SD). Multiple comparisons were evaluated by one-way analysis of variance (ANOVA) followed by Tukey s multiple comparison. All assays were performed in triplicate. A value of P < 0.05 was considered statistically significant. 3. Results 3.1. Ginsenoside Rg1 decreases cell viability To first determine the cytotoxic effect of ginsenoside Rg1 on HSC-T6 cells, we examined the cell viability when cells were treated with ginsenoside Rg1 alone or in combination with TGF-b owing to the well-characterized role in EMT in the course of fibrosis. Transforming growth factor-b displayed a profound promoting effect on cell viability with a dramatic increase in the percentage of viable cells (Fig. 1B); whereas, ginsenoside Rg1 inhibited HSC-T6 cell growth resulted in a substantial decrease in the number of survival cells in the presence or absence of TGF-b (Figs 1C and 1D). The IC 50 value of ginsenoside Rg1 in HSC-T6 cells was lg/ml. Together, these data suggest that ginsenoside Rg1 possesses a cell-killing effect toward HSC-T6 cells, which attenuates cell growth Ginsenoside Rg1 induces apoptosis Following the findings on the cell-killing effect of ginsenoside Rg1 on HSC-T6 cells, we further examined the apoptotic effect which may be attributable to the cell-killing effect. We treated HSC-T6 cells with ginsenoside Rg1 (40 lg/ml), leading to a significant apoptotic cell death, and the cells mainly went through late apoptosis (Figs. 2A and 2B). Meanwhile, the effects of ginsenoside Rg1 on the expression of Bcl-2, an antiapoptotic FIG 2 Ginsenoside Rg1 induces apoptosis. (A) Plots of HSC-T6 cell apoptosis induced by 24-h treatment of ginsenoside Rg1 (40 lg/ ml). Each quarter represents a different stage of apoptosis. (B) Bar graph showing the percentage of apoptotic HSC-T6 cells. (C) Representative blots of Bcl-2 and Bax; and actin is used as an internal control. (D) Bar graph showing the relative expression level of Bcl-2 and Bax after HSC-T6 cells were treated with 40 lg/ml ginsenoside Rg1 for 24 h. (E) Plots of HSC-T6 cell apoptosis upon the exposure to ginsenoside Rg1 (40 lg/ml) and TGF-b (1 ng/ml) alone or in combination. Each quarter represents a different stage of apoptosis. (B) Bar graph showing the percentage of apoptotic HSC-T6 cells. *P < 0.05; **P < The data were retrieved from three independent assays and analyzed by one-way analysis of variance (ANOVA) followed by Tukey s multiple comparison. 4 Ginsenoside Rg1 ameliorates liver fibrosis

5 FIG 3 Transforming growth factor-b induces a cellular EMT and fibrosis model. (A) Representative blots for collagen I, collagen III, TGF-b, Smad2/3, p-smad2/3, E-cadherin, and vimentin in HSC-T6 cells when treated with TGF-b (1 ng/ml) for 24 h; and actin is used as an internal control. (B) Representative immunofluorescent images for collagen I and E-cadherin in HSC-T6 cells when treated with TGF-b (1 ng/ml) for 24 h. E-cadherin was stained in green and nuclei were stained with DAPI in blue. Magnification: protein, and Bax, a proapoptotic protein, were examined using Western blotting assay. Ginsenoside Rg1 significantly inhibited the expression of Bcl-2, while enhancing Bax expression (Figs. 2C and 2D), which provides an explanation to its proapoptotic effect on HSC-T6 cells. Furthermore, the proapoptotic effect of ginsenoside Rg1 was tested in the presence of TGF-b which showed a promoting effect on cell growth (Fig. 1B). We showed that ginsenoside Rg1 was capable of inducing cell apoptosis in the presence of TGF-b (Figs. 2E and 2F). Collectively, these data show that ginsenoside Rg1 exerts a profound proapoptotic effect in HSC-T6 cells TGF-b induces a cellular EMT and fibrosis model Given the classical role of TGF-b in EMT, which has a close association with fibrosis, we thus established a cellular model of EMT using HSC-T6 cells upon the exposure of TGF-b. The treatment of cells with TGF-b (1 ng/ml) substantially increased the levels of collagen I and collagen III, accumulation of collagen indicates the progression of fibrosis (Fig. 3A). Also, the TGF-b/Smad signaling was examined via Western blotting assay. Transforming growth factor-b exposure dramatically increased TGF-b/Smad signal output, evident from the increase in the phosphorylation of Smad2/3 (Fig. 3A). Consequently, it FIG 4 Ginsenoside Rg1 suppresses TGF-b-induced EMT and fibrosis. (A) Representative blots for collagen I, collagen III, TGF-b, Smad2/3, p-smad2/3, E-cadherin, and vimentin in HSC-T6 cells when treated with ginsenoside Rg1 (40 lg/ml) in the presence of TGF-b (1 ng/ml) for 24 h; and actin is used as an internal control. (B) Representative immunofluorescent images for collagen I and E-cadherin in HSC-T6 cells when treated with ginsenoside Rg1 (40 lg/ml) in the presence of TGF-b (1 ng/ml) for 24 h. E-cadherin was stained in green and nuclei were stained with DAPI in blue. Magnification: Wei et al. 5

6 BioFactors FIG 5 Ginsenoside Rg1 suppresses intracellular ROS level and nuclear translocation of Nrf2. (A) Ginsenoside Rg1 decreases intracellular ROS level when HSC-T6 cells were treated with ginsenoside Rg1 (40 lg/ml) in the presence of TGF-b or H 2 O 2 for 24 h. (B) Representative blots for cytoplasmic and nuclear Nrf2 in HSC-T6 cells when treated with ginsenoside Rg1 (40 lg/ml) in the presence of TGF-b for 24 h. *P < 0.05; **P < 0.01; ***P < led to a significant decrease in the level of E-cadherin, while increasing vimentin (Fig. 3A). The immunofluorescent staining showed that TGF-b treatment increased collagen I level but decreased E-cadherin level (Fig. 3B). Taken together, HSC-T6 cells undergoe EMT and fibrosis upon the exposure to TGF-b Ginsenoside Rg1 suppresses TGF-b-induced EMT and fibrosis and intracellular ROS level In an effort to examine the effect of ginsenoside Rg1 on hepatic fibrosis, we first employed a TGF-b induced cellular EMT and fibrosis model as stated above (Fig. 3). Using HSC-T6 cell model, we observed that ginsenoside Rg1 profoundly antagonized TGF-b-induced EMT and fibrosis phenotype (Fig. 4). Ginsenoside Rg1 significantly reduced TGF-b-induced expression of collagen I and collagen III (Fig. 4A). Meanwhile, ginsenoside Rg1 substantially inhibited TGF-b/Smad signaling as shown by the decrease in phosphorylation of Smad2/3 but increase in E-cadherin level. Also, TGF-b-induced vimentin level was decreased when HSC-T6 cells were exposed to FIG 6 Rg1 ameliorates liver fibrosis in vivo. (A) Mice body weight increased when received ginsenoside Rg1 treatment. Representative H&E staining images (B) and Masson staining images (C) showing protective effects of ginsenoside Rg1 on CCl 4 -induced liver injury. (D) Representative MRI images showing ginsenoside Rg1 ameliorates liver fibrosis in vivo. (E) Ginsenoside Rg1 reduced ALT and AST levels. Magnification: *P < 0.05; **P < 0.01; ***P < The data were retrieved from three independent assays and analyzed by one-way analysis of variance (ANOVA) followed by Tukey s multiple comparison. 6 Ginsenoside Rg1 ameliorates liver fibrosis

7 ginsenoside Rg1 (Fig. 4A). In addition, immunofluorescent staining showed that ginsenoside Rg1 decreased the level of collagen I and increased E-cadherin. Besides, our data show that ginsenoside Rg1 treatment significantly reduces TGF-binduced intracellular ROS level (Fig. 5A). Notably, our Western blot data show that ginsenoside Rg1 treatment significantly increased the key antioxidant proteins regulator, Nrf2 level in nuclear compared to TGF-b treatment group, whereas decreased cytoplasmic Nrf2 level (Fig. 5B). Collectively, ginsenoside Rg1 exhibits inhibitory effects on EMT, fibrosis, ROS production, and Nrf2 nuclear translocation in HSC-T6 cells, which may be attributable to its beneficial action in liver Ginsenoside Rg1 ameliorates liver fibrosis To further interrogate the beneficial effect of ginsenoside Rg1 on hepatic fibrosis, we examined this effect in a CCl 4 -induced mice model of hepatic fibrosis. As shown in Figs. 6 and 7, ginsenoside Rg1 dramatically protected CCl 4 -induced liver injury. In comparison to CCl 4 -induced liver injury, ginsenoside Rg1 substantially attenuated the toxic effect and alleviated body weight (Fig. 6A); and protected liver from CCl 4 -induced liver damage as shown in H&E and Masson staining (Figs. 6B and 6C) and MRI examination (Fig. 6D). Consequently, these beneficial actions of ginsenoside Rg1 resulted in an ameliorated liver function. Ginsenoside Rg1 significantly reduced ALT and AST level, compared to model group. It is worth noting that ginsenoside Rg1 significantly protected liver from CCl 4 -induced fibrosis, evident from the reduction in the level of IV-C, HA, and LN (Fig. 7A). We also examined the effect of ginsenoside Rg1 on the expression of potential molecular targets involved in EMT, including TGF-b and E-cadherin, to explain the beneficial action. As shown in Fig. 7B, ginsenoside Rg1 dramatically decreased the level TGF-b and collagen, while increasing E-cadherin, suggesting that ginsenoside Rg1 may regulate TGF-b-mediated EMT signaling to exert its antifibrosis effect in liver. 4. Discussion Owing to the increasing popularity and substantial clinical consequence, liver fibrosis has caught extensive attention. There is a great deal of effort that has been devoted to elucidating the molecular mechanism and developing efficacious therapeutics for liver fibrosis. However, so far, there is no targeted therapeutic for liver fibrosis. Thus, there is an urgency to develop novel therapeutics for liver fibrosis treatment. In this study, we studied the beneficial effects and molecular mechanism of ginsenoside Rg1, a pharmacologically active component isolated from a traditional Chinese medicine Ginseng, on liver fibrosis in vitro and in vivo. We demonstrated that ginsenoside Rg1 was able to control and reverse liver fibrosis. We also studied the mechanism of action showing that FIG 7 Rg1 inhibits TGF-b-mediated EMT. (A) Ginsenoside Rg1 reduced IV-C, HA, and LN levels. (B) Representative IHC images for TGF-b, collagen I, and E-cadherin in liver tissue when mice were treated with ginsenoside Rg1 (30 and 60 mg/kg). Magnification: 2003, *P < 0.05; **P < The data were retrieved from three independent assays and analyzed by one-way analysis of variance (ANOVA) followed by Tukey s multiple comparison. Wei et al. 7

8 BioFactors ginsenoside Rg1 suppressed EMT and intracellular ROS production, at least in part, via TGF-b/Smad and Nrf2-mediated signaling pathways. A wealth of evidence suggests that EMT has a central role in liver fibrosis in response to a variety of stimuli [13], and it has been shown an association between liver fibrosis and liver carcinogenesis [10]. Thus, deciphering the regulatory network of EMT will facilitate the management of liver fibrosis. Epithelial to mesenchymal transition is associated with the loss of cell adhesion molecules and cell polarity. For example, E- cadherin is considered an active suppressor of cell invasion and growth. In addition, tight junctions that are composed of claudin and occludin proteins and form a continuous barrier function in the regulation of paracellular permeability and in the maintenance of cell polarity and block the movement of transmembrane proteins between the apical and the basolateral cell surfaces. In addition, ZO-1, 2, and 3 are peripheral membrane adaptor proteins, linking junctional transmembrane proteins such as occluding and claudin to the actin cytoskeleton [21]. All these molecules are weaving together contributing the complexity of EMT and liver fibrosis. Many studies have shown that EMT-inducing transcription factors (Twist, snail, slug, and ZEB), are involved in cell migration and invasion through different signaling cascades, for example Akt, signal transducer, and activator of transcription 3 (STAT3), MAPK, and Wnt pathways, with the ultimate consequence of decrease in E-cadherin and increase in metastatic proteins, such as N-cadherin, vimentin, and matrix metalloproteinase-2 [22]. It is worth noting that TGF-b superfamily signaling plays a critical role in the regulation of cell growth, differentiation, and development in a wide range of biological systems. In particular, the TGF-b/Smad signal has a pivotal role in EMT [13]. In this study, ginsenoside Rg1 significantly increases the level of E-cadherin, indicating an epithelial-like phenotype change; whereas, ginsenoside Rg1 can dramatically decrease the level of vimentin, which is a mesenchymal-like phenotype marker. Notably, ginsenoside Rg1 is able to tune down the TGF-b/Smad signal output by decreasing the phosphorylation level of Smad2/3, indicating ginsenoside Rg1 exerts its beneficial effects via suppressing TGF-b/Smad-mediated EMT. Besides, previously published studies showed that ginsenoside Rg1 regulated Keap1-Nrf2-ARE signaling pathway, contributing to the hepatoprotective effect [23,24]. Nrf2 is a key transcription regulator of antioxidant proteins, stimulates antioxidant proteins expression under several stress conditions. Gao et al. [23] reported that ginsenoside Rg1 reduces ROS production and interacts with Keap1 protein, and hence protects Nrf2 from Keap1-induced degradation. In line with the previous study, our data have shown that ginsenoside Rg1 suppresses intracellular ROS production with the involvement of Nrf2-mediated signaling pathway in HSC-T6 cells. Indeed, the antioxidative effect of ginsenoside Rg1 has been well documented and provides an explanation to many beneficial actions, including the cardiovascular protective effect. However, owing to the multiple targets effect, the antioxidative activity of ginsenoside Rg1 also contributes to the hepatoprotective effect. FIG 8 A schematic model of ginsenoside Rg1 effects on the liver fibrosis. 8 Ginsenoside Rg1 ameliorates liver fibrosis

9 5. Conclusions In conclusion, we showed that ginsenoside Rg1 inhibited cell proliferation and suppressed EMT in vitro and in vivo. Also, ginsenoside Rg1 ameliorated liver function and reversed liver fibrosis. The mechanism of action for the beneficial effect of ginsenoside Rg1 may be ascribed to the downregulation of TGF-b/Smad and stimulation of Nrf2-mediated signaling pathways (Fig. 8). Acknowledgements The authors appreciated the financial support from the Chongqing foundation of Basic Science and Preface Technology Research (cstc2015jcyjbx0079); and the programs of Yongchuan Hospital of Chongqing Medical University (Grant No. YJJC201704). References [1] Murphy, S. L., Xu, J., and Kochanek, K. D. (2013) Deaths: final data for Natl. Vital. Stat. Rep. 61, [2] Massey, V., Cabezas, J., and Bataller, R. (2017) Epigenetics in liver fibrosis. Semin. Liver. Dis. 37, [3] Bataller, R., and Brenner, D. A. (2005) Liver fibrosis. J. Clin. Invest. 115, [4] Weiskirchen, R., and Tacke, F. (2016) Liver fibrosis: from pathogenesis to novel therapies. Dig. Dis. 34, [5] Liu, J., and Fan, D. (2007) Hepatitis B in China. Lancet 369, [6] Tang, Y. J., Khalaf, A. T., Liu, X. M., Xu, C. X., Zhao, W., et al. (2014) Zinc finger A20 and NF-kappaB correlate with high-risk human papillomavirus of squamous cell carcinoma patients. Tumour Biol. 35, [7] Mitchell, A. E., Colvin, H. M., and Palmer Beasley, R. (2010) Institute of Medicine recommendations for the prevention and control of hepatitis B and C. Hepatology 51, [8] Davis, G. L., Alter, M. J., El-Serag, H., Poynard, T., and Jennings, L. W. (2010) Aging of hepatitis C virus (HCV)-infected persons in the United States: a multiple cohort model of HCV prevalence and disease progression. Gastroenterology 138, , 521.e1 521.e6. [9] Edmison, J., and McCullough, A. J. (2007) Pathogenesis of non-alcoholic steatohepatitis: human data. Clin. Liver Dis. 11, ix. [10] Uehara, T., Ainslie, G. R., Kutanzi, K., Pogribny, I. P., Mukshelishvili, L., et al. (2013) Molecular mechanisms of fibrosis-associated promotion of liver carcinogenesis. Toxicol. Sci. 132, [11] Brenner, D. A. (2009) Molecular pathogenesis of liver fibrosis. Trans. Am. Clin. Climatol. Assoc. 120, [12] Hernandez-Gea, V., and Friedman, S. L. (2011) Pathogenesis of liver fibrosis. Annu. Rev. Pathol. 6, [13] Zhao, Y. L., Zhu, R. T., and Sun, Y. L. (2016) Epithelial-mesenchymal transition in liver fibrosis. Biomed. Rep. 4, [14] Seton-Rogers, S. (2016) Epithelial-mesenchymal transition: untangling EMT s functions. Nat. Rev. Cancer 16, 1. [15] Davis, F. M., Stewart, T. A., Thompson, E. W., and Monteith, G. R. (2014) Targeting EMT in cancer: opportunities for pharmacological intervention. Trends Pharmacol. Sci. 35, [16] Attele, A. S., Wu, J. A., and Yuan, C. S. (1999) Ginseng pharmacology: multiple constituents and multiple actions. Biochem. Pharmacol. 58, [17] Christensen, L. P. (2009) Ginsenosides chemistry, biosynthesis, analysis, and potential health effects. Adv. Food Nutr. Res. 55, [18] Yu, J., Liu, X., Ke, C., Wu, Q., Lu, W., et al. (2017) Effective suckling C57BL/ 6, Kunming, and BALB/c mouse models with remarkable neurological manifestation for Zika virus infection. Viruses 9, 165. [19] Pereira, N. B., Campos, P. P., de Jesus Oviedo Socarras, T., Pimenta, T. S., Parreiras, P. M., et al. (2012) Sponge implant in Swiss mice as a model for studying loxoscelism. Toxicon 59, [20] Yang, D. H., Ye, Z. Y., Jin, B., He, X. J., Zhang, Q., et al. (2011) Salvianolate inhibits cytokine gene expression in small intestine of cirrhotic rats. World J. Gastroenterol. 17, [21] Nauseef, J. T., and Henry, M. D. (2011) Epithelial-to-mesenchymal transition in prostate cancer: paradigm or puzzle? Nat. Rev. Urol. 8, [22] Tania, M., Khan, M. A., and Fu, J. (2014) Epithelial to mesenchymal transition inducing transcription factors and metastatic cancer. Tumour Biol. 35, [23] Gao, Y., Chu, S., Shao, Q., Zhang, M., Xia, C., et al. (2017) Antioxidant activities of ginsenoside Rg1 against cisplatin-induced hepatic injury through Nrf2 signaling pathway in mice. Free Radic. Res. 51, [24] Gao, Y., Chu, S., Zhang, Z., and Chen, N. (2017) Hepataprotective effects of ginsenoside Rg1 a review. J. Ethnopharmacol. 206, Wei et al. 9

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