Polyguluronate sulfate (PGS) attenuates

International Journal of Biological Macromolecules 114 (2018) 592–598

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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Polyguluronate sulfate (PGS) attenuates immunological liver injury in vitro and in vivo Yanyun Gao a, Wei Liu a, Wei Wang a,b,⁎, Xia Zhao a,b,⁎, Fahe Wang c a Key Laboratory of Marine Drugs, Ministry of Education, Shandong Provincial Key laboratory of Glycoscience and Glycoengineering, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China b Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China c State Key Laboratory of Bioactive Seaweed Substances, Qingdao Brightmoon Seaweed Group Co Ltd., Qingdao 266400, China

a r t i c l e

i n f o

Article history: Received 27 December 2017 Received in revised form 14 March 2018 Accepted 19 March 2018 Available online 21 March 2018 Keywords: Polyguluronate sulfate Con A Liver injury Oxidative stress Hepatoprotective effect

a b s t r a c t Hepatocyte damage, especially immunological liver injury, is a key process in the pathogenesis of hepatitis virusinduced liver diseases. The aim of this study was to investigate the protective effects of polyguluronate sulfate (PGS) against immunological liver damage. The results showed that PGS significantly reduced the H2O2-induced oxidative stress and increased the cell viability in HepG2 hepatocytes. PGS also suppressed the production of malondialdehyde (MDA), lactate dehydrogenase (LDH), TNF-α, and IL-6, while up-regulating the activity of SOD in HepG2 cells. Further, PGS (150 and 300 mg/kg/day) significantly attenuated the elevation of serum glutamate pyruvate transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBiL), in addition to liver MDA and NO levels in Con A-induced immunological liver injury within mice (P b 0.05). Significant improvements of organ indexes (liver, spleen, and thymus) were observed in PGS-treated mice. PGS also significantly reduced the disorganization of hepatocytes and decreased the inflammatory cell infiltration caused by Con A treatment, suggesting that PGS was able to attenuate Con A-induced liver injury. In conclusion, PGS possesses significant hepatoprotective effects on immunological liver injury in vitro and in vivo, and this may be related to its antioxidant activities. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Hepatitis B is a major global health problem caused by hepatitis B virus (HBV) and results in acute and chronic hepatitis that may progress to liver cirrhosis and hepatocellular carcinoma (HCC) [1,2]. Liver injury that is observed after chronic hepatitis B virus infection is mainly mediated by the immune response against the virus [3]. The Concanavalin A (Con A)-induced immunological liver injury model established by Tiegs et al. [4] in mice has been widely used as a model for human viral hepatitis and autoimmune hepatitis. Con A is a plant lectin and T-cell mitogen that rapidly induces T-helper (Th) cell activation, primarily of CD4 + T-cells, that then leads to inflammatory liver injury in mice [4,5]. Thus, the Con A-induced hepatitis model may also be used to identify effective therapeutic agents for hepatitis B that exhibit low side-effects including liver damage [6,7]. Polyguluronate sulfate (PGS) is a low molecular weight sulfated polysaccharide (Fig. 1A) that is prepared by chemical sulfation of α-

⁎ Corresponding authors at: School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China. E-mail addresses: [email protected], (W. Wang), [email protected]. (X. Zhao).

https://doi.org/10.1016/j.ijbiomac.2018.03.098 0141-8130/© 2018 Elsevier B.V. All rights reserved.

1,4-poly-L-guluronic (PG) acid that is isolated from alginate. Our previous research showed that PGS possessed anti-HBV activities in HepG2.2.15 cells [8]. However, little is known regarding the effect of PGS on immunologic liver injury in vitro and in vivo. In this study, a H2O2-treated HepG2 cell model was used to evaluate the protective effects of PGS against oxidative stress damage. The Con A-induced hepatitis model [9] was then used to explore the hepatoprotective effects of PGS against Con A-induced liver injury. 2. Materials and methods 2.1. Chemicals and reagents PGS was prepared in our lab [10]. Con A and 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St Louis, MO, USA). Bifendate Pills (BP) were obtained from Wanbang Pharmaceutical Co., Ltd. (Zhejiang, China), and dissolved in 0.9% saline. Serum aspartate aminotransferase (AST), glutamate pyruvate transaminase (ALT), and total bilirubin (TBiL) enzyme-linked immunosorbent assay (ELISA) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). The lipid peroxidation malondialdehyde (MDA) assay kit, total superoxide dismutase (SOD) assay kit, lactate dehydrogenase (LDH) release assay kit, nitric

Y. Gao et al. / International Journal of Biological Macromolecules 114 (2018) 592–598

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Fig. 1. Chemical structure and characterization of PGS. (A) The structure of PGS. (B) The HPGPC chromatogram of PGS. (C) The FT-IR spectrum of PGS. (D) The 13C NMR spectrum of PGS.

oxide (NO) assay kit, and enhanced bicinchoninic acid (BCA) protein assay kit were obtained from the Beyotime Institute of Biotechnology (Nantong, China). ELISA IL-6 and TNF-α assay kits were purchased from Dakewe Biotech Co., Ltd. (Shenzhen, China). Dulbecco's Modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco (Grand Island, NY, USA). All other reagents that were used were analytical grade.

2.2. Cells and animals HepG2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were maintained in DMEM medium supplemented with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. Cells were incubated at 37 °C in an atmosphere of 5% CO2. Male Kunming (KM) mice weighing 20 ± 2 g were housed under identical conditions at 22 ± 2 °C in a 12-h light-dark cycle with free access to food and water. All of the procedures involving animals were conducted in compliance with the guidelines approved by the Animal Care and Use Committee of the School of Medicine and Pharmacy at the Ocean University of China (Qingdao, China).

2.3. Cell cytotoxicity and viability assays The cytotoxic effects of PGS against HepG2 cells were evaluated using the MTT assay [11]. Briefly, after 48 h of drug treatment, culture media was replaced with 100 μL of PBS containing MTT (at a final concentration of 0.5 mg/mL) and incubated at 37 °C for 4 h. Then, 200 μL of dimethyl sulfoxide (DMSO) was added after removal of the supernatant, and the absorbance values at 570 nm were measured with a microplate reader (Bio-Rad, USA). For antioxidant stress experiments, HepG2 cells were exposed to H2O2 at specific concentrations with or without PGS (50–200 μg/mL) treatment for 8 h at 37 °C. Cell viability of HepG2 cells was then evaluated with the MTT assay [11]. Cell viability is expressed as a percentage of the non-treated control.

2.4. Detection of MDA, SOD, LDH, TNF-α, and IL-6 in HepG2 cells HepG2 cells were exposed to 4 mmol/L H2O2 with or without PGS (50–200 μg/mL) treatment for 8 h at 37 °C. The relative contents of LDH and MDA in the cellular supernatants and the levels of SOD in cell

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followed by Tukey's tests, while considering a P value b 0.05 as statistically significant.

lysates were measured using commercially available kits according to the manufacturer's instructions. The levels of IL-6 and TNF-α in the cellular supernatants were measured by ELISA kits according to the manufacturer's protocol.

3. Results and discussion

2.5. Con A-induced liver injury model

3.1. Characterization of PGS

The immunological liver injury model was established by intravenous injection of Con A (20 mg/kg) via the tail vein of mice [12]. The mice were randomly divided into six groups (10 mice per group), including the normal control group, model control group (injected Con A only), BP-treated (standard reference) group (150 mg/kg/day) [13], and three PGS-treated groups (75, 150, and 300 mg/kg/day). PGS was introduced by intragastric administration at 75, 150, or 300 mg/kg/day for seven consecutive days. The control groups were given equal volumes of 0.9% saline. On the seventh day, 4 h after gavage, Con A injections were given to all of the groups except for the normal control group, which was given 0.9% saline. Injection dosages into the tail veins were 0.1 mL/20 g for all groups. No food was provided after Con A injection, but water was available ad libitum for the following 8 h.

The average molecular weight of PGS was 9.1 kDa as determined by high performance gel permeation chromatography (HPGPC) [10] and the purity N98% (Fig. 1B). The degree of polymerization (DP) of PGS was 20–30 (Fig. 1A). The intrinsic viscosity, [η] of PGS was 9.3 dL/g at the concentration of 1% (w/w) according to the method previously described [16]. In the FT-IR spectrum (Fig. 1C), the PGS were characterized with two absorption pecks at 1232 and 840 cm−1 derived from S_O and C\\O\\S bond stretching, respectively. The results indicated that PG was successfully sulfated. The structure of PGS was further determined by nuclear magnetic resonance spectroscopy (NMR) analysis. In the 13C NMR spectrum of PGS (Fig. 1D), the peaks at 173.76, 99.37,

A 120

2.6. Blood and tissue collection

*

80 Cell viability

Mice were killed 8 h after Con A injection. Blood was then withdrawn from the eye socket, and let stand for 1 h at room temperature (RT) before centrifugation at 3000 rpm for 10 min at 4 °C. The liver, spleen, and thymus of mice were promptly removed and their weights were recorded to calculate organ indexes.

100

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2.7. Organ indexes

0 Control 62.5

2.8. Determination of serum ALT, AST, and TBiL levels Serum AST, ALT, and TBiL levels in blood samples were determined using ELISA kits according to the manufacturer's instructions.

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Concentration(µg/mL)

B 120 100 Cell viability(%)

The organ indexes [14] of the mice liver, spleen, and thymus were calculated by the following equations: Liver index = [liver weight (g) / mice weight (g)] × 100. Spleen index = [spleen weight (g) / mice weight (g)] × 100. Thymus index = [thymic weight (g) / mice weight (g)] × 100.

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80 60 40 20 0 Control 0.25 0.5

2.9. Determination of liver MDA and NO contents

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Concentration of H2 O2 (mmol/L)

2.10. Liver histology For histological investigation [15], liver tissues were removed from a portion of the left lobe, fixed in 10% formalin, and embedded in paraffin. After pathological sectioning and hematoxylin & eosin (HE) staining, liver histopathologic changes were examined by light microscopy. A total of 18 tissue sections were analyzed. 2.11. Statistical analysis All of the experimental data are expressed as means ± S.D., and statistical significance was calculated using GraphPad Prism 5.0. Comparison between groups was performed using one-way ANOVA analysis

C 120 100 Cell viability(%)

Liver tissues were homogenized using PBS in an ice bath, and the ratio of liver tissue weight to homogenate volume was 10% (w/v). MDA and NO levels were determined using an MDA assay kit and a NO detection kit, respectively. The concentration of total protein in the lysates was quantified with an enhanced BCA protein assay kit. All of the procedures were performed according to the manufacturer's instructions.

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0 H2O2(4mM) PGS( g/mL)

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Fig. 2. The cytotoxicity assay of PGS and its effects on H2O2-mediated oxidative stress in HepG2 cells. (A) Cytotoxicity of PGS in HepG2 cells. HepG2 cells were incubated with PGS at indicated concentrations for 48 h. Then, cell viability was measured by the MTT method. Values are means ± S.D. (n = 6). (B) HepG2 cells were incubated with different concentrations of H2O2 (0.25, 0.5, 1, 2, 4, 8, and 16 mmol/L) for 8 h. (C) HepG2 cells were treated with or without different concentrations of PGS (50, 100, and 200 μg/mL) in combination with 4 mmol/L of H2O2 for 8 h. Then, cell viability was measured by the MTT method. Values are means ± SD (n = 6). ##P b 0.01 vs. the normal control group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. the H2O2-treated control group.


A

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H2 O2 (4mM) PGS( g/mL)

SOD (% of control)

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3.3. Influence of PGS on antioxidant markers in HepG2 cells

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LDH (% Cytotoxicity)

H2O2-treated HepG2 cell model was used to evaluate the protective effects of PGS against oxidative stress-induced liver damage in vitro. The cytotoxic effects of PGS against HepG2 cells were first determined by MTT assay. As shown in Fig. 2A, the maximum non-toxic concentration of PGS was about 250 μg/mL. The CC50 (50% cytotoxicity concentration) value for PGS was 1728 μg/mL. This result was used to determine the dosage range of PGS for the subsequent experiments. The survival rate of the HepG2 cells was negatively correlated with the concentration of H2O2 (Fig. 2B). The cell viability of the HepG2 cells was 49.6% when the concentration of H2O2 was 4 mmol/L, indicating that the oxidative damage model was successfully established (Fig. 2B). To determine the protective effects of PGS on H2O2-induced cell death, an MTT assay was performed in hepatocytes after being treated with 4 mmol/L H2O2 for 8 h in the presence or absence of PGS (50– 200 μg/mL). As shown in Fig. 2C, treatment with PGS (100 or 200 μg/ mL) significantly increased the cell viability of the HepG2 cells as compared to the H2O2-treated control group (P b 0.05). Thus, PGS may be able to protect hepatocytes against oxidative stress.

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C

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There is a balanced status between oxidation and antioxidation in normal organisms, and SOD is known as the main antioxidase that plays an important role in such a balance [18]. MDA is closely related to lipid peroxidation, and LDH is associated with hepatitis and cardiomyopathies. Thus, MDA, SOD, and LDH were chosen as antioxidant markers in this study. As expected, treatment with 4 mmol/L of H2O2 for 8 h significantly increased the levels of MDA and decreased the contents of SOD as compared to the normal control group (P b 0.01) (Fig. 3A and B). However, treatment with PGS (100 and 200 μg/mL) significantly decreased the levels of MDA (Fig. 3A) and increased the contents of SOD in the HepG2 cells (P b 0.05) (Fig. 3B). Moreover, treatment with PGS (50, 100, or 200 μg/mL) significantly reduced the H2O2-induced LDH

A

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Fig. 3. Effects of PGS on the activities of antioxidant markers in HepG2 cells. HepG2 cells were treated with or without different concentrations of PGS (50, 100, and 200 μg/mL) in combination with 4 mmol/L of H2O2 for 8 h. The relative contents of MDA (A) in the cell supernatants and SOD (B) in the cell lysates were determined, respectively. (C) LDH release in the culture media was determined. Results are expressed as a percentage of a cell lysis control provided by the manufacturer. Results are presented as means ± SD of three independent experiments. ##P b 0.01 vs. the normal control group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. the H2O2-stimulated control group.

and 77.79 ppm that were assigned to the resonances of C-6, C-1, and C4, respectively. In addition, signals at 71.51, 67.26, and 65.61 ppm were assigned to the resonances of C-3, C-5, and C-2, respectively. The signal at 74.03 ppm for sulfated C-3 and the signal at 69.45 ppm for sulfated C2 were observed, respectively, supporting that the sulfation occurred at the C-3 and C-2 positions of PG [10]. The intensity of signal C-2S was almost the same as that of signal C-3S, indicating that there was no obvious selectivity for the C2-OH and C3-OH in the sulfation reaction. By calculating the signal intensities of the C2S/C2 and C3S/C3, it was found that about 81% and 78% of the C2-OH and C3-OH groups were replaced by sulfate groups in PGS, namely, the substitution degree of sulfate in PGS was about 1.6. 3.2. Effects of PGS on oxidative stress-induced hepatocyte injury in vitro H2O2 possesses strong oxidizing properties and can cause deleterious physiological effects by inducing lipid peroxidation and DNA damage [17], which is a major mediator of oxidative stress. Herein, the

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Fig. 4. Effects of PGS on the production of inflammatory cytokines in HepG2 cells. After treatment with or without PGS (50, 100, and 200 μg/mL) in H2O2-treated HepG2 cells, the relative contents of IL-6 (A) and TNF-α (B) in the cell culture supernatants were determined by using ELISA kits, respectively. Results are presented as means ± SD of three independent experiments. ##P b 0.01 vs. the normal control group; ⁎⁎P b 0.01 vs. the H2O2-stimulated control group.

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Table 1 Effects of PGS on organ indexes against Con A induced liver injury in mice. Groups

Liver index

Spleen index

Thymus index

Normal control Model control BP (150 mg/kg/day) PGS (75 mg/kg/day) PGS (150 mg/kg/day) PGS (300 mg/kg/day)

4.87 ± 0.55 6.99 ± 0.48## 5.6 ± 0.6⁎⁎ 6.7 ± 0.26 5.93 ± 0.72⁎⁎ 5.62 ± 0.57⁎⁎

0.53 ± 0.11 1.08 ± 0.21## 0.67 ± 0.07⁎⁎ 1.07 ± 0.15 0.82 ± 0.1⁎⁎ 0.62 ± 0.09⁎⁎

0.32 ± 0.06 0.23 ± 0.02## 0.31 ± 0.06⁎ 0.26 ± 0.05 0.27 ± 0.04 0.31 ± 0.05⁎

Data are expressed as mean ± SD in each group (n = 10). ## P b 0.01 vs. normal control group. ⁎ P b 0.05. ⁎⁎ P b 0.01 vs. model group.

release (P b 0.05) (Fig. 3C). These results suggested that PGS may be able to regulate the antioxidant markers to protect HepG2 cells from death.

3.4. Effects of PGS on inflammatory cytokines in vitro IL-6 and TNF-α are common inflammatory cytokines in hepatocyte injury that can be induced by various stimuli. As shown in Fig. 4A and B, the expression levels of the two inflammatory cytokines in the HepG2 cells were significantly elevated by H2O2 treatment and they were 1.58- and 1.82-fold higher than those in the non-treated hepatocyte cells (P b 0.01), respectively. After treatment with PGS (100 or 200 μg/mL) combined with 4 mmol/L of H2O2 for 8 h, the secretion levels of TNF-α and IL-6 were decreased significantly as compared to those in the H2O2-treated control cells (P b 0.01) (Fig. 4A and B). Therefore, PGS may also be able to inhibit the inflammatory responses induced by oxidative stress.

3.5. Effects of PGS on organ damage in Con A-treated mice The Con A-induced immunological liver injury mouse model was established by the intravenous injection of Con A (20 mg/kg) via the tail vein of mice [15,19]. Organ indexes, including the liver, spleen, and thymus indexes, were first evaluated in Con A-treated mice with or without drug treatment. As shown in Table 1, after Con A treatment, the liver and spleen indexes were significantly increased while the thymus index was significantly decreased as compared to those in the normal control group (P b 0.01). The thymus and spleen indexes are associated with cellular immunity and humoral immunity, respectively, and may reflect the functional states of experimental mice [20]. After pre-treatment with PGS (150 or 300 mg/kg/day) for seven days, the liver and spleen indexes were significantly decreased as compared to that in the Con A-treated model group (P b 0.01) (Table 1). PGS (300 mg/kg/day) treatment also significantly increased the thymus index (P b 0.05), comparable to that in the BP-treated group (150 mg/kg/day) (Table 1). A dose-effect correlation was observed in the PGS-treated groups in regard to the decrease of the spleen and liver indexes, and the increase of the thymus index was induced by the Con A-treatment (Table 1). Therefore, PGS may be able to attenuate the organ damage caused by Con A treatment in the immunological liver injury model. 3.6. Effects of PGS on serum ALT, AST, and TBiL levels in Con A-treated mice The fulminant hepatitis symptoms, including inflammatory infiltration, hepatocyte nucleus rupture, and the elevation of ALT and AST levels in serum, may be present in the Con A-induced liver injury mice [4]. Herein, the serum ALT, AST, and TBiL levels truly significantly increased in the mice after Con A treatment as compared to those in the normal control group (P b 0.01) (Fig. 5), suggesting that the

A 15 TBiL (µmolL -1)

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Normol Model BP(150) 75 Groups

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Karmen unit

ALT AST

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PGS(mgkg -1 d-1 )

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0 Normol Model BP(150) 75

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PGS(mg kg d ) Groups Fig. 5. Effects of PGS on serum ALT, AST, and TBiL levels against Con A-induced liver injury in mice. The TBiL (A), ALT, and AST (B) levels in serum were detected in Con A-treated mice by ELISA assay, respectively. Data are expressed as mean ± SD in each group (n = 10). ##P b 0.01 vs. the normal control group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. the model control group.

Fig. 6. Effects of PGS on liver MDA and NO levels on Con A-induced liver injury in mice. The MDA (A) and NO (B) levels in the liver were detected in Con A-treated mice, respectively. Data are expressed as mean ± SD in each group (n = 10). ##P b 0.01 vs. the normal control group; ⁎P b 0.05, ⁎⁎P b 0.01 vs. the model control group.


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Fig. 7. Histopathological changes in Con A-induced mice livers. The liver tissues were excised from normal (A), Con A-injured control (B), BP-treated (150 mg/kg/day) (C), PGS-treated (75 mg/kg/day) (D), PGS-treated (150 mg/kg/day) (E), and PGS-treated (300 mg/kg/day) (F) mice. The liver sections were stained with hematoxylin and eosin, and examined by light microscopy. Representative photographs are presented at a magnification of ×400. Scale bar represents 20 μm.

immunological liver injury mouse model was established successfully. After the oral administration of PGS (150 or 300 mg/kg/day) for seven days before Con A treatment, the serum ALT, AST, and TBiL levels were significantly decreased as compared to those in the model group (P b 0.05) (Fig. 5A and B). A PGS treatment (300 mg/kg/day) provided the best protective effects among the three PGS groups, which was comparable to the effects of BP treatment (150 mg/kg/day) (Fig. 5). These results suggested that PGS had a protective effect against Con A-induced liver injury in mice. 3.7. PGS inhibited the elevation of MDA and NO levels in con A-treated mice To further explore the protective effects of PGS against Con A-induced liver injury, MDA and NO levels in the liver were also evaluated by ELISA assay. As shown in Fig. 6, the liver MDA and NO levels were significantly increased in the mice after Con A treatment as compared to those in the normal control group (P b 0.01). However, the oral administration of PGS (150 or 300 mg/kg/day) for seven days before Con A treatment significantly reduced the liver MDA and NO levels as compared to the model control group (P b 0.05) (Fig. 6). It has been reported that liver injury after Con A treatment may induce the expression of NOS (iNOS and NOS2), which is able to generate large amounts of NO [21]. MDA, the final product of lipid peroxidation, can seriously damage the cell membrane structure and cause cell swelling and necrosis, which reflects the extent of liver injury [22]. Therefore, these results suggested that the protective effects of PGS against Con A-induced liver injury in mice may be related to its antioxidant activities.

cells with obvious hepatic sinusoids, a prominent nucleus, and visible central veins (Fig. 7A). In contrast, in the Con A-treated model group, the hepatic sinusoids stenosis had almost disappeared, the erythrocyte siltation and vacuolization had emerged, and the infiltration of inflammatory cells and the rupture of the hepatocyte nucleus were observed in the liver sections (black arrow, indicated in Fig. 7B). However, PGS treatment (150 or 300 mg/kg/day) significantly reduced the disorganization of the hepatocytes and decreased the infiltration of inflammatory cells caused by Con A treatment (Fig. 7E and F) as compared to the effects of BP treatment (150 mg/kg/day) (Fig. 7C). In summary, these results clearly indicated that PGS had a significant protective effect against Con A-induced liver injury in mice. Considering that PGS also possesses protective effects against H2O2-induced oxidative stress in hepatocytes, we hypothesize that the hepatoprotective effects of PGS on immunological liver injury, both in vitro and in vivo, may be largely related to its antioxidant activities. 4. Conclusions Our results indicated that PGS significantly attenuated liver injury symptoms and reduced the elevation of serum ALT, AST, TBiL, and liver MDA and NO levels in the Con A-treated mice. PGS also inhibited H2O2-induced oxidative stress and increased cell viability in the HepG2 cells. Thus, PGS possesses hepatoprotective effects on immunological liver injury, both in vitro and in vivo, and they may be related to its antioxidant properties. Considering the anti-HBV activities of PGS in HepG2.2.15 cells [8], PGS is worthy of further investigation as a potential alternative or a complementary therapy for HBV infection.

3.8. Histopathological examination of Con A-treated mice livers Acknowledgements To directly evaluate the protective effects of PGS against liver injury caused by Con A treatment, the histopathological analysis of liver tissues was also performed [23]. Briefly, the liver sections were prepared and stained with HE after drug treatment (Fig. 7). The histology of the liver sections in the normal control group showed as normal hepatic

This work was supported by the NSFC-Shandong Joint Fund (U1606403, U1706210), the Innovation Project of Qingdao National Laboratory for Marine Science and Technology (No. 2015ASKJ02), the National Natural Science Foundation of China (31500646, 81741146),

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