The Mechanisms Of The Fucoidan From Sea Cucumber Apostichopus Japonicus On Attenuating Lipopolysaccharide-challenged Liver Injury In C57BL/6J Mice

Inflammation is a defensive response of the body to external stimuli,and many diseases are often accompanied by inflammatory reactions.Furthermore,inflammation is strongly associated with many pathological conditions including cardiovascular diseases,cancers,and neurological diseases.Cyclo-oxygenase inhibitors,such as acetylsalicylic acid,are commonly used anti-inflammatory drugs.But long-term use of these can cause serious toxic side effects,such as gastric erosions and kidney damage.Fucoidan（FUC）is the main component of the body wall of sea cucumber.As a natural low-toxic water-soluble substance,it has a variety of biological activities,such as anti-inflammatory,anti-cancer,and anti-coagulation.This study was designed to preparation of the FUC from the sea cucumber A.japonicus（Aj-FUC）and investigate whether Aj-FUC has an anti-inflammatory effect in the liver of lipopolysaccharide（LPS）-challenged C57BL/6J mice.In this study,the crude polysaccharide was isolated from the sea cucumber A.japonicus by papain degradation（pH=6）.Then the crude polysaccharide was fractionated using ?KTA-FPLC pure 150 protein purification system combined with Q-Sepharose^TM Fast Flow and Sephacyl S-200 H.R Columns.The monosaccharide composition,purity and molecular weight of the Aj-FUC were determined by high performance liquid chromatography（HPLC）and gel permeation chromatography（GPC）,respectively.Fucolysated chondroitin sulfate（FCS）and FUC were separated from the crude polysaccharide sea cucumber A.japonicus by QFF column taking 0² mol/L of NaCl as eluent.The molecular weight,sugar content and sulfate content of Aj-FUC was 202.3 kDa,64.9%,and 19.8%,respectively.HPLC results showed that Aj-FUC was mainly composed of mannose,glucosamine,glucuronic acid,galactosamine,galactose,and fucose,in a molar ratio of 3.9:1.0:2.6:7.8:1.7:23.6.Twenty-five C57BL/6J mice（male,23±4 g）were purchased from Beijing HFK Bioscience Co.,Ltd.（license number:SCXK2014-0004）.Mice were free to access of water and chow diet.After acclimatization for 7 days,the mice were randomly divided into five groups,the blank control group（n=5,normal saline）,the model group（n=5,normal saline+LPS）,the aspirin group（n=5,20 mg/kg/d+LPS）,the low-dose Aj-FUC group（n=5,Aj-L,60mg/kg/d+LPS）and the high-dose Aj-FUC group（n=5,Aj-H,180 mg/kg/d+LPS）.Mice were treated for 7 days with normal saline or different drugs by gavage as described above.Two hours after the last administration,foods were taken away and the mice were challenged with LPS（5.0 mg/kg）or normal saline by intraperitoneal injection.Four hours later,the mice in each group were sampled,and the plasma and liver were kept for the rest of the study.Western blot（WB）,real-time quantitative PCR（RT-PCR）,and immunohistochemistry（IH）were used to detect the expression of inflammatory factors and related signaling pathways.In the present study,our results demonstrated that the Aj-FUC treatment significantly reduced the protein expression of tumor necrosis factor（TNF-α）and interleukin-6（IL-6）,and the mRNA expression of interleukin-1β（IL-1β）and inducible nitric oxide synthase（iNOS）compared to the model group.Further study showed that the Aj-FUC treatment remarkably decreased the phosphorylation of Nuclear factorκB p65（NF-κB p65）compared to the model group,especially in the Aj-L group.Of note,Aj-FUC intervention significantly reduced the phosphorylation of p38 and extracellular-signa1regulated kinase（ERK1/2）compared to the model group,especially in the Aj-L group,but it has no significant effect on c-Jun N-terminal kinase（JNK）.Furthermore,we used the MILLIPLEX MAP kit to test the protein kinase B/mammalian target of rapamycin（AKT/mTOR）signaling pathway.Our data showed that the Aj-FUC significantly inhibited the phosphorylation mTOR,p70 ribosomal protein S6 kinase（p70S6K）,ribosomal protein S6（RPS6）,and glycogen synthase kinase-3β（GSK-3β）compared to the model group,but no glycogen synthase kinase-3α（GSK3α）.In addition,we used IH method to further confirm the effects of the Aj-FUC on the expression of TNF-α,NF-κB p65,p38,and ERK1/2,and the results were consistent with that of WB.Collectively,our date indicated that Aj-FUC can reduce inflammation in the liver of LPS-challenged C57BL/6J mice via down-regulating MAPK/NF-κB and AKT/mTOR pathways.