β-glucoside Purified from Black Rice Protects Mice against Hepatic

Jun 14, 2015 - against Hepatic Fibrosis Induced by Carbon Tetrachloride via. Inhibiting Hepatic Stellate Cell Activation. Xinwei Jiang,. †. Honghui ...
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Cyanidin-3‑O‑β-glucoside Purified from Black Rice Protects Mice against Hepatic Fibrosis Induced by Carbon Tetrachloride via Inhibiting Hepatic Stellate Cell Activation Xinwei Jiang,† Honghui Guo,§ Tianran Shen,† Xilan Tang,† Yan Yang,*,† and Wenhua Ling*,†,‡ †

Department of Nutrition, School of Public Health, Sun Yat-Sen University, Guangzhou 510080, People’s Republic of China Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Guangzhou 510080, People’s Republic of China § Department of Nutrition, Henry Fok School of Food Science and Engineering, Shaoguan University, Shaoguan 512005, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: This study investigated whether cyanidin-3-O-β-glucoside (Cy-3-G), a predominant anthocyanin, could exert a protective role on liver injury and its further mechanisms of the anti-fibrosis actions in mice. The results demonstrated that the treatment of Cy-3-G (800 mg/kg diet) for 8 weeks significantly attenuated hepatotoxicity and fibrosis in carbon tetrachloride (CCl4) administered mice. Cy-3-G strongly down-regulated the expression of α-smooth muscle actin (α-SMA), desmin, and matrix metalloproteinase (MMPs), which showed its suppression effect on the activation of hepatic stellate cells (HSCs). In addition, Cy-3-G favorably regulated oxidative stress and apoptosis in liver. Furthermore, Cy-3-G ameliorated the infiltration of inflammatory cells such as neutrophils and leukocytes and meanwhile suppressed the production of pro-inflammatory cytokines and growth factors. In conclusion, daily intake of Cy-3-G could prevent liver fibrosis progression in mice induced by CCl4 through inhibiting HSC activation, which provides a basis for clinical practice of liver fibrosis prevention. KEYWORDS: cyanidin-3-O-β-glucoside, anthocyanins, liver fibrosis, hepatic stellate cells



INTRODUCTION Liver injury is currently a prevalent disease, which is caused by either acute or chronic cellular injury due to various reasons, such as toxic chemicals, viral hepatitis B or C, alcohol abuse, and nonalcoholic steatohepatitis.1 With the continued existence of injury, the pathological progression of liver could cause modulation of hepatic architecture and portal hypertension, which finally lead to liver fibrosis, liver failure, and even cancer.2 Hepatic fibrosis results from the wound-healing response of the liver to repeated injury. When the liver is suffering from acute injury, parenchymal cells renew and replace the apoptotic or necrotic cells; however, if an attack such as oxidative stress continues, it will lead to the persistent hepatic injury, accompanied by the regeneration of hepatocytes fails, and excessive extracellular matrix (ECM) replaces the dead cells. Hepatic stellate cells (HSCs) are the key cells producing ECM in the liver. Under chronic damage in the liver, HSCs undergo a phenotypic transformation from quiescent retinoidstoring cells to proliferative, fibroblast-like cell type.3 Activated HSCs migrate to the sites of damaged tissue, present a phonotype of high expression of α-smooth muscle actin (αSMA) and desmin, secreting type I and III collagen, and finally ECM accumulates and liver fibrosis is formed.4 Activation of the HSCs can be provoked by a series of reactions; it is initiated by the injury of hepatocytes exposed to lipid peroxides, and then the products from damaged hepatocytes and cytokines, including pro-inflammatory cytokines, growth factors (TGF-β and PDGF), and chemokines from Kupffer or other inflammatory cells, irritate the activation of HSCs.5 © XXXX American Chemical Society

Because of the vast clinical burden of liver injury and its overlapping pathogenesis with cardiovascular disease (CVD) and diabetes, ongoing research and knowledge about its treatment and prevention are advancing rapidly.6 However, various side effects of antifibrotic therapy have been gradually discovered, and suitable treatment remains a major concern. Previous studies have shown that phytochemicals in plant food could be a group of effective materials to benefit liver function.7 Thus, further studies on daily phytochemical intake to prevent liver injury progressing is valuable, and this may shed light on fibrosis reversing mechanisms as well as proof of guidance for healthy diet. Anthocyanins (ACNs) are glycosylated polyhydroxy, watersoluble flavonoid pigments, naturally existing in colorful fruits, vegetables, and grains, so that we have plenty of chances to ingest abundant ACNs during our daily diets.8 ACNs can reduce cellular oxidative stress by their antioxidant effects.9 Previous studies showed that ACNs could have protection effects on cardiovascular disease,10 obesity, and diabetes.11,12 The protective function of ACNs was also studied in liver diseases, and it was revealed that anthocyanin-rich extracts attenuate nonalcoholic steatohepatitis and associated fibrosis in mice; 13 this kind of antifibrosis effect was found in dimethylnitrosamine- 14 or carbon tetrachloride-induced15,16 liver injury in animal models as well. However, the majority of Received: April 30, 2015 Revised: June 13, 2015 Accepted: June 13, 2015

A

DOI: 10.1021/acs.jafc.5b02181 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry the studies have focused on ACN-rich foods14,16 or beverages,17 which contain multiple compounds and different concentrations or subclasses of ACNs. Furthermore, the rates of absorption and elimination as well as the relative bioavailability of ACNs present in different foods in vivo are different.18 Therefore, it is important to examine the direct effects of pure ACNs on liver injury to elucidate their mechanisms of action in vitro and in vivo.19,20 However, to the best of our knowledge, the direct role of the purified monomer form of ACNs in liver injury remains unclear, and their effects on liver fibrosis in vivo has not been systematically studied. In the present study, we aimed to explore whether cyanidin3-O-β-glucoside (Cy-3-G), the most abundant monomer form of ACNs in plants,21 could protect against liver damage caused by carbon tetrachloride (CCl4), as well as suppress the paths of the activation of HSCs.



Histological Study and Immunohistochemistry. Liver samples were separated and fixed in 10% neutral buffered formalin and then embedded in paraffin, sectioned to a thickness of 5 μm. Then the slides were performed with hematoxylin and eosin (H&E) staining for general observation or with Sirius Red for collagen fiber detection. Under a light microscope (Leica, Solms, German), inflammatory cell infiltration in hepatic lobules and necrosis were observed. Then, the inflammation activity were evaluated and divided into four parts: portal area inflammation (P), lobular inflammation (L), patch necrosis (PN), and bridging necrosis (including lobular necrosis, BN). Every item was recorded as 1, 2, 3, or 4 on the basis of degrees of pathological changes. The score counting formula was P + L + 2 × (PN + BN).22 The percentage of fibrosis regions in the liver was measured on five high-power (200×) fields per slide and then calculated by Image-pro Plus 6.0. The observer was blinded to the group distribution when the analyses were made. Immunostaining was performed on paraffin section. For the test of the activation of hepatic stellate cells, desmin was visualized with Alex488 conjugated goat anti-rabbit IgG secondary antibody, and DAPI was used for nuclear staining. After the slides had been incubated with anti-F4/80 or anti-CD45 at 4 °C overnight, they were treated with labeled polymer horseradish peroxidase (HRP) secondary antibody for 1.5 h, followed by DAB staining. Positive cells were counted on five high-power (200×) fields per slide. Measurements of MDA, SOD, and GSH. Liver tissue homogenates were prepared to measure hepatic malondialdehyde (MDA) following the commercial kit’s instructions (Jiancheng Biotech, Nanjing, China). Superoxide dismutase (SOD) and glutathione (GSH) were measured by the commercial kits from Beyotime (Shanghai, China). Measurements of ROS and Apoptosis. The frozen liver samples were cut into 10 μm sections and fixed in paraformaldehyde. After being washed in phosphate buffer saline (PBS) three times, the slides were incubated with DHE (5 mmol/L) in a dark condition for 30 min at 37 °C for redundant reactive oxygen species (ROS) detection. DHE can be oxidized by superoxide and changed to ethidium bromide, which then binds to the DNA of the nucleus, and red fluorescence was detected through the fluorescence microscope (Leica, Solms, Germany) under the 580 nm exciting light.23 Apoptosis of cells in the liver was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) staining (Beyotime) according to the manual; the fluorescence was detected at 510 nm through a fluorescence microscope (Leica). Thereafter, the intensity of the fluorescence was analyzed by Image-pro Plus 6.0. Protein Extraction and Western Blotting. Proteins were extracted from liver tissue, using RIPA lysis buffer (Beyotime) and 1% PMSF and 1% protease inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged at 12000 g for 15 min, and the supernatants were used for immunoblotting; meanwhile, the total protein concentration of the tissue homogenates was determined by BCA protein assay kit (Thermo, Rockford, IL, USA). Protein extractions were separated by using SDS-PAGE on 10% polyacrylamide gels and transferred to 0.45 μm polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking for 2 h with 5% skimmed milk in TBS-T buffer (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20), the membrane was incubated with primary antibodies against α-SMA or GAPDH at 4 °C for overnight. The membrane was incubated with HRP-conjugated secondary antibodies and using ECL Reagent Kit (Thermo) to make the film exposed. The band densities were quantified using an image analyzer, Quantity One System (BioRad, Richmond, CA, USA). All protein quantifications were adjusted for the corresponding GAPDH level. RNA Extraction and Quantitative Real-Time PCR. Total RNA was extracted from liver tissue with Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Then total RNA was reverse-transcribed to complementary DNA (cDNA) using the Reverse Transcription System by the cDNA Synthesis kit (Takara, Dalian, China). Quantitative real-time PCR was performed with SYBR Green Supermix kit (Takara) according to the manufacturer’s instructions and played on a Vii7 system (ABI, Carlsbad, CA, USA).

MATERIALS AND METHODS

Cy-3-G Extraction and Analyses. Black rice (Oryza sativa L. indica) was obtained from Guangdong province, China. ACNs were extracted from black rice following our previous methods.10 Then, high purity Cy-3-G was isolated from ACNs by the method of medium-pressure liquid chromatography (MPLC). After analysis by HPLC-PAD, the purity of Cy-3-G was >96.5%. The detailed methods are shown in the Supporting Information. Reagents and Antibodies. Antibody for α-smooth muscle actin (α-SMA) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and for glyceraldehyde phosphate dehydrogenase (GAPDH) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-desmin and anti-F4/80 antibodies were purchased from Abcam (Cambridge, UK); anti-CD45 antibody and Alex-488 conjugated goat anti-rabbit IgG secondary antibody were purchased from Goodbio Technology (Wuhan, China). All other secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Reagents for immunohistochemistry, such as hydrogen peroxide and diaminobenzidine (DAB), were obtained from Zhongshan Jinqiao Biotechnology (Beijing, China). 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Nantong, China). Fluorescent probe dihydroethidium (DHE) was from Calbiochem (San Diego, CA, USA). All other chemicals used in this study were of analytical grade from Sigma-Aldrich or Invitrogen unless otherwise noted. Animals and Diets. This study was carried out in agreement with the recommendations of the Guide for the Care and Use of Laboratory Animals. All animal procedures were approved by the Animal Care and Protection Committee of Sun Yat-Sen University. SPF, male, C57BL/ 6 mice (8 weeks old) were purchased from the SYSU animal center (Guangzhou, China). The animals were maintained at 25 °C with a 12 h light/dark cycle and ad libitum access to water and chow. Mice were randomly divided into three groups (each with eight mice), including a control group, a CCl4-treated group, and a Cy-3-G plus CCl4 group. CCl4-treated mice were given an intraperitoneal (ip) injection of 10% CCl4 in olive oil (0.5 μL pure CCl4/g BW) twice weekly for 8 weeks, whereas the control group received vehicle only. At the same time, Cy3-G (800 mg/kg diet) was added to standard diet, which was supplied for the Cy-3-G plus CCl4 group; the other groups of mice lived on standard diet. All of the animals were sacrificed 72 h after the last dose of CCl4 or vehicle. After narcotizing the mice by pentobarbital sodium, blood was collected by extirpating eyeballs for serum biochemistry analysis. Livers were excised, partly fixed for histological analysis, and the rest was frozen in liquid nitrogen and then stored at −80 °C until analyzed. Determination of Serum Transaminase Levels and Cytokines. Serums were separated from blood by centrifuging under 4 °C, at 3000 g for 15 min, then alanine and aspartate aminotransferases (ALT and AST) were measured by an assay kit from Jiancheng Biotech (Nanjing, China) according to the manual. Serum cytokines (TNF-α, TGF-β, and IL-17) were analyzed by ELISA technique using commercial kits from eBioscience (San Diego, CA, USA). B

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Journal of Agricultural and Food Chemistry Relative gene expression was calculated using the 2−ΔΔCt method. Primers used in the test are shown in the Supporting Information. Statistical Analysis. SPSS 16.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis, and all results are presented as the mean ± SEM. Data were analyzed using Student’s t test (for two groups), oneway ANOVA, and Dunnett’s test (for three groups). Mann−Whitney’s rank sum test was used to calculate the statistical significance of the pathological scores of inflammation. P < 0.05 was considered statistically significant. Graph bars were obtained using Graph Pad Prism 5.0 software (San Diego, CA, USA).

peroxidation, elevated sharply after the treatment of CCl4 (Figure 3B). However, Cy-3-G treatment significantly inhibited the oxidative stress levels of ROS and MDA (P < 0.05). Glutathione (GSH) is an antioxidant, which can prevent damage from reactive oxygen species. After the treatment of CCl4, the level of GSH significantly decreased (Figure 3C). With the supplementation of Cy-3-G, the level of GSH tends to recover, but without significant difference compared to the CCl4 group (P = 0.3102). Antioxidant enzymes can protect cellular compounds against damage induced by free radicals. In the present study, total SOD was detected by the WST-1 assay. CCl4 treatment caused a severe decrease of SOD level (P < 0.05), whereas addition of Cy-3-G to the diet ameliorated changes of the hepatic SOD activity significantly (Figure 3D). Cy-3-G Ameliorated Apoptosis of Hepatic Cells. ALT and AST have been recognized as biomarkers of liver injury.24 The activities of serum ALT and AST were respectively increased 92 and 83% in CCl4-treated mice compared to the control group (Figure 4A,B). However, the treatment of Cy-3G showed an inhibition of this elevation; it significantly ameliorated the level of ALT (P < 0.05) and decreased the level of AST (P = 0.159). Apoptotic cells in the liver were detected by TUNEL staining (Figure 4C). Dying cells, with DNA damage, can be combined to the fluorescent dye (cyanine 3) by the catalysis of terminal deoxynucleotidyl transferase. Thereafter, cyanine 3 release red fluorescence under excitation of 510 nm light. After treatment with CCl4, the apoptotic cells in the liver increased significantly. Although the number of apoptotic cells in the Cy-3-G group showed no significant differences compared to the CCl4 group, it showed a trend of decrease (P = 0.088). Cy-3-G Suppressed Leukocyte Recruitment but Had No Significant Effect on the Activation of Kupffer Cells. Administration of CCl4 caused hepatocyte injury and followed with more infiltrating inflammatory cells. To investigate periportal inflammation in the liver, we performed immunostaining with anti-CD45 (Figure 5A), which was a biomarker of leukocytes.25 In the CCl4 group, positive cells increased nearly 30-fold (Figure 5B), compared to control animals. Adding Cy3-G to the diet significantly alleviated the number of CD45+ cells compared to the CCl4 group. Kupffer cells are resident macrophages in liver, which were activated by damage amplification and recruited from the bloodstream.26 Upon activation, Kupffer cells release proinflammatory cytokines involving TNF-α and fibrogenic factors such as TGF-β1 and PDGF, which will stimulate HSCs to be activated.27 F4/80 is a specific marker of activated Kupffer cells; after the treatment with CCl4, F4/80 positive cells increased almost 13-fold compared with the control group (Figure 5C,D). Interestingly, Cy-3-G treatment had no significant effect compared with the CCl4 group. Cy-3-G Attenuated the Level of Pro-inflammation Cytokines and Increased the Level of Anti-inflammation Cytokine. Pro-inflammation cytokines are released from both injured hepatocytes and nonparenchymal cells, which play important roles in driving fibrogenesis. In our present study, TNF-α, IL-1α, and IL-17 were detected. After the treatment with CCl4, TNF-α levels in the serum and tissue (Figure 6A,B) were increased nearly 1-fold compared to the control group, whereas they significantly decreased after treatment with Cy-3G. However, detected by the qRT-PCR method, IL-1α had no significant changes in the CCl4 or CCl4 plus Cy-3-G (Figure 6C) groups. Previous studies have shown that monocyte



RESULTS General Parameters and Histopathological Analysis. During the experiment period, mice with CCl4 treatment showed a trend of reducing body weight and increasing relative liver weight compared to the control mice without statistical significance as shown in Table 1. Table 1. Data from General Parameters Evaluated in Different Groupsa initial body wt (g) final body wt (g) food consumption (g/mouse/day) relative liver wt (%)

control

CCl4

CCl4 + Cy-3-G

26.05 ± 0.62 28.52 ± 0.73 2.74 ± 0.08

26.11 ± 0.79 28.00 ± 0.78 2.83 ± 0.12

26.39 ± 0 0.52 27.43 ± 0.44 2.67 ± 0.13

3.96 ± 0.13

4.24 ± 0.12

4.10 ± 0.29

a

Body and relative liver weights were examined in the mice model. Results are expressed as mean ± SEM (n = 8 mice/each). Comparisons between all groups were evaluated using one-way analysis of variance (ANOVA).

To estimate the protective function of Cy-3-G on liver injury, H&E staining and Sirius staining were carried out on liver tissue slides (Figure 1). CCl4-treated groups developed liver damage with the performance of hepatocellular lesions, inflammation, and liver fibrosis. Whereas Cy-3-G ameliorated the degree of patch necrosis, bridging necrosis and less inflammatory cells (mainly neutrophils) infiltrated into the portal area; fibrillar collagen sediment was rarely found in the portal/periportal area. The inflammatory scores were calculated (Figure 1C; Table 2), and Sirius Red staining for the fibrosis was evaluated (Figure 1D); Cy-3-G ameliorated both scores (P < 0.05). Therefore, according to histopathological analysis, Cy-3-G showed a protective effect on liver injury and fibrosis. Cy-3-G Depressed the Activation of HSCs. HSCs are cores in the progression of liver injury. Their activation, accompanied by cytokine release and production of matrix molecules, proteases, and their inhibitors, finally leads to liver fibrosis. We investigated the suppressing effect of Cy-3-G on the activation of HSCs induced by CCl4. As shown in Figure 2A, immunoblotting (left panel) and qRT-PCR (right panel) analysis revealed that α-SMA was less expressed in the Cy-3-G group compared to the CCl4 group. In addition, desmin as another marker of the activated HSCs (Figure 2B,C) was less detected by immunostaining in the Cy-3-G-treated group compared with the CCl4 group. Moreover, matrix metalloproteinase (MMP), such as MMP-2 and MMP-9, mRNA levels sharply increased in the CCl4 group, whereas they were significantly decreased by the intervention of Cy-3-G (Figure 2D,E). Cy-3-G Attenuated Oxidative Stress and Increased Antioxidant. Hepatic levels of ROS increased significantly (Figure 3A), and MDA, the terminal product of lipid C

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Figure 1. H&E staining and Sirius staining on liver tissue: (A) hematoxylin and eosin stain (the inflammation score was evaluated and calculated (C)); (B) Sirius Red staining (quantitative analysis of Sirius Red staining was carried out by using Image Pro-Plus (D)). All slides are based on the examination of five different fields, 200× magnifications. Results are expressed as the mean ± SEM; (∗) P < 0.05, significantly different compared to the control group; (#) P < 0.05, significantly different compared to the CCl4 group. n = 6 (control group) or 7 (other groups).

Table 2. Pathological Inflammatory Scores in Liver Tissuesa group

P+L+2× (PN + BN)

S0

S1

S2

S3

S4

P

control CCl4 CCl4+Cy-3-G

0.667 ± 0.083 2.643 ± 0.280 1.750 ± 0.299

5 0 1

1 1 3

0 4 3

0 2 0

0 0 0

0.001* 0.017#

7A,C). Interestingly, Cy-3-G had no significant effect on the level of TGF-β1 in serum (Figure 7B). Monocyte chemotactic protein (MCP-1) is a kind of typical chemokine expressed in various cells in the liver, which in general promotes the migration of fibrogenic cells to the site of liver injury.30 The relative mRNA expression of MCP-1 dropped nearly 1-fold after Cy-3-G intervention compared to the CCl4 group (Figure 7D).

The score of inflammation was given by P + L + 2 × (PN + BN) (P, portal area inflammation; L, lobular inflammation; PN, patch necrosis; BN, bridging necrosis). n (control): n(CCl4):n(CCl4+Cy-3-G) = 6:7:7. Mann−Whitney’s rank sum test was used to calculate the statistical significance: (*) P < 0.05 compared to the control group; (#) P < 0.05 compared to the CCl4 group. a



DISCUSSION In our present study, we demonstrated that in CCl4-treated mice, Cy-3-G extracted from black rice attenuated liver injury and fibrosis. Furthermore, we found that addition of Cy-3-G suppressed the activation of hepatic stellate cells, which may result from the amelioration of liver oxidative stress, hepatocyte apoptosis, and inflammation induced by CCl4. To our knowledge, this is the first study showing the positive effects of Cy-3-G on CCl4 injury mouse model and uncovers the possible paths of restraining activation of HSCs. ACN extractions from plant foods were proved to have a series of benefits on various chronic diseases, but it is difficult to explain the biological mechanism of ACNs, because the food extractions usually contain an enormous variety of bioactive compounds. Through 2009, more than 600 kinds of ACNs had been extracted and identified from plants, such as Cy-3-G, Dp3-G, and Pg-3-G.31 Therefore, it is useful and valuable to study one composition of anthocyanin to elucidate the biological effects and the mechanism of actions. Cyanidin exists in about 50% of all ACNs in fruits and vegetables. Moreover, Cy-3-G is the most common anthocyanin with a glycoside.32 In the

production of TNF-α was a critical downstream response to the IL-17 produced by Th17 cells.28 The level of IL-17 was detected in both serum and liver tissue by ELISA; IL-17 elevated 1-fold in serum and 0.5-fold in liver tissue after the treatment with CCl4, which were significantly decreased by Cy3-G supplementation (Figure 6D,E). IL-10 is a kind of antiinflammatory cytokine. In our present study, Cy-3-G treatment enhanced the relative mRNA expression of IL-10, which is suggestive of promotion of anti-inflammation cytokine (Figure 6F). Cy-3-G Treatment Decreased Liver Growth Factors and MCP-1. HSCs are an important source of growth factors in the liver, but also respond to these factors. TGF-β1 and PDGF are two potent growth factors that stimulate activation and proliferation of HSCs.29 The elevated levels of TGF-β1 and PDGF mRNA expression in the liver induced by CCl4 were suppressed significantly by treatment with Cy-3-G (Figure D

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Figure 2. Cy-3-G attenuates the activation of HSCs in liver: (A) α-SMA protein (left panel) and mRNA (right panel) levels were evaluated; (B) desmin was detected by immunofluorescence and then qualified by the fluorescent intensity in every field (C); relative expressions of hepatic MMP2 and MMP-9 mRNA levels were measured by RT-PCR (D, E). Values are expressed as the mean ± SEM, n = 5; (∗) P < 0.05 compared to the control group; (#) P < 0.05 compared to the CCl4 group.

The present study showed that supplementation of Cy-3-G has great effects on suppressing the liver fibrosis induced by CCl4. Hepatic fibrogenesis is a complex pathogenesis responding to various toxic stimuli. HSC activation is considered to play a key role in fibrotic progression. Activated HSCs were marked with higher expression of α-SMA, more desmin, and generation of collagen type I. In the present study, these elevated markers resulting from CCl4 injection were significantly decreased with the treatment of Cy-3-G. Furthermore, MMP-2 and MMP-9 mRNA levels were significantly suppressed by Cy-3-G. It is universally recognized that MMPs are a family of zinc metalloendopeptidases, which may contribute to the regression of liver fibrosis through cleavage of the fibrillar extracellular matrix. On the other hand, MMPs are promptly expressed by activated HSCs in response to diverse hepatic injury and are considered to promote disease progression.38 Thus, MMPs play a dual role

present study, high-purity Cy-3-G as a monomer of anthocyanin was extracted from black rice and purified by MPLC, which has many merits including lower cost, higher speed, and greater loading of samples compared with the other purification methods.33 Cy-3-G is nontoxic, and a moderate dose was adopted (800 mg/kg diet) for the present study according to previous researches in vivo.34−36 It has been reported that CCl4 increases free radicals, which further causes lipid peroxidation of cellular and organelle membranes; thus, it is considered as one of the most widely used toxic agents to induce liver injury in animals.37 We used a very low dose of CCl4 (0.5 μL/g BW) injection for 8 weeks, which caused a moderate degree of liver injury according to histological analysis (Figure 1). Under this situation, we could investigate the preventive effects of Cy-3-G on liver fibrosis. E

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Figure 3. Cy-3-G attenuated oxidative stress and increased antioxidant defense system in CCl4 administered mice: (A) liver ROS level detected by dihydroethidium (DHE) staining (representative photomicrographs (100×) are shown and mean of red fluorescence density of per field quantified by ImageJ (right panel)); (B) MDA, lipid peroxidation assessed in liver homogenate; (C) hepatic GSH and (D) SOD measured by commercial assay kit (n = 6−7, mean ± SEM; (∗) P < 0.05 versus control; (#) P < 0.05 versus CCl4).

in liver fibrosis, depending on the timing of their production. The early temporary high expression of MMPs may destroy the surrounding tissue in order to deposit newly synthesized ECM.39 In our present study, Sirius Red staining of liver collagens showed the liver fibrosis occurring at an early stage of the animal model, and MMP-2 and MMP-9 expression significantly increased in the CCl4 group. Because of the early stage of liver fibrosis, MMPs are considered as markers of the activation of HSCs. Cy-3-G could decrease MMP-2 and MMP9 expression, suggesting that Cy-3-G may suppress the activation of HSCs. As is well-known, CCl4 in vivo is metabolized by the cytochrome P450 system to highly reactive free radicals and reactive oxygen species, such as trichloromethyl (CCl3) and trichloromethyl peroxyl (CCl3O2), which initiate lipid peroxidation and generate MDA.16 Subsequently, elevated ROS attack hepatocytes, which leads to apoptosis.40 By the treatment with Cy-3-G, ROS level and oxidative products such as MDA were significantly decreased and antioxidative SOD level increased to catalyze the dismutation of superoxide to hydrogen peroxide and oxygen. Nonetheless, GSH as a nonenzymatic defense substance slightly changed in the CCl4 and Cy-3-G group, which may account for the balance between GSH and glutathione disulfide (GSSG) being restored efficiently at this

early phase of liver injury, and the enzyme glutathione reductase was still available to bring GSSG back to GSH.40 Thus, Cy-3-G showed no significant effect on GSH. ALT and AST were both considered sensitive indicators of hepatocyte integrity. It was revealed that either acute or chronic injury to the liver would eventually result in an increase of aminotransferases.41 Furthermore, apoptosis is responsible for the physiological removal of unwanted cells, involving damaged cells.42 We found that the treatment with Cy-3-G significantly diminished the liver apoptosis accompanied by the decreasing AST and ALT levels in circulation. These results indicated that Cy-3-G protective effects on hepatocytes may be related to the reduction of oxidative stress in the liver, similar to a previous report that ACNs or Cy-3-G could protect high glucose or methionine and choline deficiency caused by mouse hepatocyte apoptosis by modulating mitochondrial dysfunction and the PI3K/Akt pathway.34,43 The generation of oxidative stress together with the injured hepatocytes could increase tissue inflammation and drive disease progression more intensely.44 This is reflected in our finding that CCl4 up-regulated inflammation degree in the liver, whereas Cy-3-G treatment could attenuate liver inflammation significantly. To evaluate liver inflammation, a liver biopsy assessment system was employed to evaluate the inflammation F

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Figure 4. Cy-3-G alleviated apoptosis of hepatic cells. ALT (A) and AST (B) levels in the circulation were evaluated. Cell apoptosis in the liver tissue was detected by TUNEL assay (100×), and positive cells were counted in every field (C). Results are expressed as the mean ± SEM; (∗) P < 0.05 significantly different compared to the control group; (#) P < 0.05 versus the CCl4 group.

Figure 5. Effects of Cy-3-G on the inflammatory cells recruitment in CCl4-treated mice. Immunostaining was performed on liver tissue sections. (A) Anti-CD45 was used to detect the infiltration of leukocytes in liver, and (B) F4/80 antibody was used to identify the activated Kupffer cells (magnification 200×). Five typical fields per slide were randomly examined, and then the numbers of positive cells in every field were counted and analyzed (C, D). Values are expressed as the mean ± SEM, n = 5; (∗) P < 0.05 compared to the control group; (#) P < 0.05 compared to the CCl4 group.

score in our experiments.22 As our present study showed, Cy-3G could decrease neutrophils and attained a lower liver inflammation score (Figure 1; Table 2). Furthermore, it reduced liver CD45 positive cells, which were leukocytes, infiltrated to the periportal zone as inflammation cells (Figure 5). We also studied Kupffer cells, resident liver macrophages, which were also activated during liver injury. Activated Kupffer cells are enabled to launch biochemical attack and initiate interactions with HSCs by releasing a variety of biologically active mediators.26 Interestingly, in our study, the results revealed that Cy-3-G had no significant suppressing effect on

the activation of Kupffer cells in CCl4-treated mice, which seems to be incompatible to previous protective effects on ROS or hepatocyte apoptosis. This phenomenon may be due to the early stage of liver injury and the weak effect of Cy-3-G on Kupffer cells. With the progression of liver injury, pro-inflammatory cytokines, growth factors, and chemokines are released, which subsequently stimulate the activation of HSCs and migration. Not only are TNF-α, TGF-β, PDGF, and other cytokines produced by Kupffers but also infiltrating neutrophils, macrophages, or HSCs.45 We identified that Cy-3-G could restrain G

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Figure 6. Cy-3-G ameliorated inflammatory cytokines induced by CCl4 treatment in mice (A) TNF-α and (C) IL-17 in serum and liver tissues measured by ELISA assay. IL-1α (B) and (D) IL-10 mRNA in liver tissue were analyzed by qRT-PCR. Values are expressed as the mean ± SEM, n = 5; (∗) P < 0.05 compared to the control group; (#) P < 0.05 compared to the CCl4 group.

Figure 7. Cy-3-G decreased growth factors and MCP-1 in CCl4-treated mice. TGF-β in liver tissue (A) and serum (B) were measured by ELISA assay. PDGF (C) and MCP-1(D) mRNA levels in liver tissue were analyzed by qRT-PCR. Values are expressed as mean ± SEM, n = 5; (∗) P < 0.05 compared to the control group; (#) P < 0.05 compared to the CCl4 group.

(Scheme 1). Cy-3-G can be partly absorbed into circulation48 and may have an opportunity to react with HSCs. Moreover, Cy-3-G was investigated to prevent HSC proliferation and collagen synthesis in vitro.20 In conclusion, Cy-3-G was able to attenuate liver injury and prevent fibrosis in CCl4-treated mice. Moreover, Cy-3-G could reduce liver oxidative stress, alleviate apoptosis of hepatic cells, suppress liver inflammation, and finally restrain the activation of HSCs. Our study implied that regular intake of Cy-3-G in the daily diet could prevent the progression of hepatic fibrosis and be a potent element to treat liver injury. However, further studies are needed to fully understand the cellular and molecular mechanisms of Cy-3-G on HSCs.

the level of TNF-α, TGF-β, PDGF, and MCP-1 and increase the level of IL-10; the anti-inflammation effects are similar to the previous study about Cy-3-G on obesity, which might result from decreasing NF-κB activity.46 Furthermore, we detected IL17, which was significantly down-regulated by Cy-3-G in both liver and circulation. IL-17 is mainly secreted from Th17 cells that can promote tissue inflammation by induction of other pro-inflammatory mediators and recruitment of leukocytes to the site of inflammation.47 In our present study, a Cy-3-G antiinflammatory effect was evidenced by restraining inflammation cell recruitment and inflammatory cytokines in liver injury. The paracrine and autocrine sources of cytokines will finally cause the activation of HSCs. It is assumed that Cy-3-G restrains HSC activation through two possible paths; the first was to suppress the path of generating inflammatory cytokines; the second might be to directly suppress the activation of HSCs H

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interleukin-17; TGF-β, tissue growth factor-β; PDGF, plateletderived growth factor; MMP-2, matrix metalloproteinases-2; MMP-9, matrix metalloproteinases-9; NAFLD, nonalcoholic fatty liver disease; NAS, NAFLD activity score

Scheme 1. Cy-3-G Could Restrain HSC Activation Possibly by Two Paths: The First Was To Suppress the Path of Generating Inflammatory Cytokines; The Second Might Directly Suppress the Activation of HSCs





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ASSOCIATED CONTENT

* Supporting Information S

Methods of Cy-3-G extraction and analysis from black rice (Supporting Information and Methods, Figure S1 and S2) and primer sequences for quantitative real-time PCR (Table S1). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02181.



REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*(Y.Y.) Phone: 86-20-87330687. Fax: 86-20-87330446. E-mail: [email protected]. *(W.L.) Phone: 86-20-87331597. Fax: 86-20-87330446. Email: [email protected]. Author Contributions

Xinwei Jiang and Wenhua Ling were in charge of the experimental design, implementing, and preparing for the manuscript. Tianran Shen and Xilan Tang participated in mouse feeding and treatment and helped with data analysis. Yan Yang and Honghui Guo helped with biochemical analysis and modification of the manuscript. Funding

This study was supported by grants from the National Key Basic Research Program of China (973 Program, No. 2012CB517506) and National Natural Science Foundation of China (No. 81130052, 81372994). Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS Lili Yang, Huilian Zhu, and Min Xia are acknowledged for supporting the research work with suggestions. ABBREVIATIONS USED Cy-3-G, cyanidin-3-O-β-glucoside; CCl4, carbon tetrachloride; ACNs, anthocyanins; HSC, hepatic stellate cells; ECM, extracellular matrix; ROS, reactive oxygen species; ALT, alanine aminotransferase; AST, aspartate aminotransferase; α-SMA, αsmooth muscle actin; TNF-α, tissue necrosis factor-α; MCP-1, monocyte chemotactic protein-1; IL-1α, interleukin-1α; IL-17, I

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