Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 06/21/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

Page 1 of 16

Fucoidan Reduces Inflammatory Response in a Rat Model of Hepatic Ischemia–Reperfusion Injury LI Xiao-jing 1(PHD), YE Qi-fa 1* (MD) 1

Transplant Medical Engineering Research Center，Third Xiangya Hospital，Central South University,

Hunan, China *

Author for correspondence:

YE Qi-fa Tongzipo Road 138,Yuelu District Changsha 410013 China Tel: +8618501373829 Email: [email protected]

Running Title: Fucoidan reduces hepatic I/R Injury

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 06/21/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

Page 2 of 16

Abstract Ischemia and reperfusion (I/R) injury after liver transplantation is a major cause of severe complications leading to graft dysfunction. Fucoidan, a complex of sulfated polysaccharides derived from marine brown algae, has shown its anti-apoptosis as well as potential anti-inflammatory properties in previous studies. Fucoidan has also shown protective effects on I/R injured kidney and heart. However, whether Fucoidan can attenuate hepatic I/R injury has not been examined. To clarify the role of Fucoidan on hepatic I/R injury, Sprague Dawley rats were subjected to sham operation, or ischemia followed by reperfusion with treatment of saline or Fucoidan (50,100,200 mg/kg/day). The Fucoidan group showed decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels compared with the control group. Myeloperoxidase (MPO), malondialdehyde (MDA) activity and mRNA level of CD11b in Fucoidan group was significantly decreased. Hepatocellular swelling/necrosis, sinusoidal/vascular congestion and inflammatory cell infiltration were also attenuated in Fucoidan group. Expressions of TNF-α, IL-6, IL-1β, CXCL-10, VCAM-1 and ICAM-1 were markedly decreased in the samples from Fucoidan group. Fucoidan largely prevented inflammatory signaling pathway activation compared with the control group. In summary, Fucoidan can protect liver from I/R injury through suppressing inflammatory signaling pathways activation, inflammatory mediators’ expression and inflammatory cell infiltration. Key words Fucoidan; Ischemia-Reperfusion injury; Inflammation; ROS Abbreviations I/R ischemia and reperfusion; ALT alanine aminotransferase; AST aspartate aminotransferase; MPO myeloperoxidase; MDA malondialdehyde; ROS reactive oxygen species;

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 06/21/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

Page 3 of 16

1 Introduction Hypovolemic shock or surgical procedures, such as liver resection and transplantation, influence the liver blood supply dramatically. During these procedures, hepatic ischemia reperfusion (I/R) injury is a major contributor to tissue damage and deteriorate clinical outcome (Baskin-Bey et al. 2007). Moreover, hepatic I/R injury is magnified under the condition of non-alcoholic fatty liver disease (NAFLD), which is becoming a major health problem due to the obesity endemic (Reiniers et al. 2014). Hepatic I/R injury have been demonstrated characterized by activation of nuclear factor-kappa B (NF-kB) in the liver promoting proinflammatory cytokine and adhesion molecule synthesis. These result in oxygen-derived free radical production and neutrophil recruitment, further contributing to cellular injury (Serracino-Inglott et al. 2001). Inflammatory response was considered to be the key issue in causing hepatocellular damage and liver dysfunction (Jaeschke 2006; Yamanouchi et al. 2007). In NAFLD, fat-laden hepatocytes are damaged by chronic oxidative/nitrosative stress. Hepatic I/R injury could acutely exacerbate the hepatocytes damage and consequently give rise to necrotic cell death (Reiniers et al. 2014). Therefore, it has been proved that treatments focused on suppressing inflammatory response and apoptosis triggered by I/R would result in lighter hepatic injury and better consequence (Bignotto et al. 2009; Rajesh et al. 2007). Fucoidan is a group of natural fucose-containing sulfated polysaccharides enriched in brown seaweed, common seafood in East Asian. Recent researches identified Fucoidan as a collection of natural molecular with p a spectrum of bioactivities such as antioxidation, anti-inflammation, and anticoagulation (Cumashi et al. 2007; Richard et al. 2006). Moreover, previous studies have indicated Fucoidan could protect against renal I/R injury via inhibition of the MAPK signaling pathway (Chen et al. 2013; Manzo-Silberman et al. 2011). However, it has not been examined whether Fucoidan can regulate hepatic inflammation or attenuate hepatic I/R injury(Fondevila et al. 2003). Our study investigated whether Fucoidan exerted a protective role against acute hepatic I/R injury and its related mechanisms. Our experiment showed Fucoidan down regulated inflammatory response and prevented hepatic I/R injury in a dose dependent way, which indicates that Fucoidan may indicate a therapeutic candidate for preventing or treating hepatic I/R injury. 2 Materials and Methods 2.1 Materials Sprague–Dawley rats weighing 240-250 g (Joint Venture SIPPR BK Experimental Animal Co., Shanghai, China) were fed ad libitum with laboratory chow and housed in a SPF environment. Animal study was approved by the Animal Care Committee of the Xiangya School of Medicine and performed in accordance with our institutional guidelines. Fucoidan was isolated from L. japonica commercially cultured in Qingdao, China, as described previously (Zhang et al. 2003). Fucoidan was dissolved in physiological saline for animal treatment.

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 06/21/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

Page 4 of 16

2.2 Determining the Optimal Dose of Fucoidan to Protect Against Hepatic I/R Injury Using the rats liver I/R model described below, the optimal dose of Fucoidan was determined. Rat were treated with saline, 50, 100 and 200 mg/kg/d Fucoidan orally 7days before ischemia induction, respectively. After 6 h, animals were sacrificed and the serum ALT levels were examined. 2.3 Liver I/R Injury Model and Experimental Groups Rats were anesthetized with an intraperitoneal (i.p.) injection of pentobarbital (45–50 mg/kg) and placed supine on a heating pad to maintain their body temperature during surgery. Animals were subjected to a midline abdominal incision, which was kept moist with a sterile wet cotton gauze pad. All structures in the portal pedicle (hepatic artery, portal vein and bile duct) to the left and median liver lobes were occluded with an atraumatic clamp for 60 min. Reperfusion was initiated by removal of the clamp. This model caused about 70 % liver ischemia without intestinal congestion. Five milliliters of sterile saline (37 °C) was infused to the abdominal cavity before closure of the abdomen to compensate for the volume loss. In sham group, rat underwent the same protocol without vascular occlusion. In Fucoidan group, rats were fed with Fucoidan at the optimal dose 7 days before ischemia. In vehicle-treated (Saline) group, rats received saline alone. A serial of rats (5 rats/group) and the others (5 rats/group) were sacrificed 6 and 24 h after reperfusion respectively to obtain blood and damaged hepatic samples. 2.4 Biochemical Examination Blood was drawn from the postcava and centrifuged at 3,000 x g for 10 min. Serum was collected and stored at ‑20˚C. Levels of ALT and AST in the serum were measured with an autobiochemical analyzer (Hitachi, Tokyo, Japan). Liver samples were obtained from the median lobe, washed in cold saline and quickly placed into ‑196˚C liquid nitrogen during sampling and maintained at ‑80˚C until use. Prior to detection, the samples were first unfrozen at 4˚C and subsequently homogenized in ice-cold phosphate buffer (pH 7.4). The homogenates that were centrifuged at 3,500 x g for 10 min at 4˚C were used to determine the level of malondialdehyde (MDA) and myeloperoxidase (MPO), and were assessed with a spectrophotometer at 532 and 460 nm, respectively. 2.5 Liver Histological Examination Liver samples were fixed with 10 % formalin, embedded in paraffin, and cut to 4 lm thick sections. The sections were stained with hematoxylin and eosin (H&E) and histological severity of I/R injury was assessed using SUZUKI’s method (Suzuki et al. 1993). In this classification, sinusoidal congestion, hepatocytes necrosis and ballooning degeneration were graded from 0 to 4. No necrosis, congestion or ballooning was given a score 0, and severe congestion, ballooning degeneration or lobular necrosis 60 % was given a value 4. 2.6 SYBR Green Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by NEW YORK UNIVERSITY on 06/21/15 For personal use only. This Just-IN manuscript is the accepted manuscript prior to copy editing and page composition. It may differ from the final official version of record.

Page 5 of 16

Total RNA was extracted from hepatic tissue samples using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instruction. After removal of potentially contaminating DNA with DNase I (Invitrogen, USA), RNA extracted from each sample was used for RT-PCR (Mbi Fermentas Inc., Burlington, USA) to generate first-strand complementary DNA (cDNA), which was subsequently used for real time PCR. PCR was conducted using the following parameters: 50 °C for 2 min, 95 °C for 2 min, and 40 cycles of 95°C for 15s and 60°C for 30s on an ABI StepOneTM real-time RT-PCR system (Applied Biosystems, USA). The amount of mRNA for each gene was normalized to the amount of mRNA for β-actin in each sample, and relative expression was calculated. Relative mRNA level of each gene in the sham group samples was adjusted to 1 and served as a calibrator. Hence, steady-state mRNA levels were expressed as an n-fold difference relative to the calibrator. 2.7 Western Blotting Liver tissues were homogenized in ice-cold lysis buffer, and the supernatants from homogenates were used for Western blotting. Protein concentration was determined with BCA Protein Assay Kit (Pierce) according to the manufacturer’s instruction. Samples with equal amounts of protein were mixed with sodium dodecyl sulfate(SDS)- loading buffer, and then boiled for 10 min, sonicated for 30 s, and centrifuged at 10,000 g for 10 min. Resulting supernatants (30 lg protein per lane) were separated by 12 % SDS–polyacrylamide gels electrophoresis. Protein was electro-transferred to nitrocellulose membranes with transblot system (Bio-Rad, Hercules, CA), then was blocked for 2 h at room temperature with TBST (Tris- buffered saline, pH 7.6, containing 0.05 % Tween 20) containing 5 % non-fat milk. After washed twice with TBST, the membranes were incubated overnight at 4 °C with the primary antibodies against JNK, p38 mitogen-activated protein kinase (MAPK), ERK, phospho-JNK, phospho-p38 MAPK and phospho-ERK (Cell Signaling, MA, USA). The bound antibodies were detected by horseradish peroxidase-conjugated anti-rabbit or rat IgG antibodies. Quantification was performed by densitometric analysis. 2.8 Statistical Analysis All data were expressed as mean ±SD. Results were analyzed by one-way ANOVA, and Student’s t test was used for judging statistical significance if differences were established. P