Research On The Protective Effect And Mechanism Of Crude Dictyophora Rubrovalvata Polysaccharide On Alcoholic Liver Injury In Rats

Objective:The purpose of the research is to explore the protective effect and mechanism of crude Dictyophora rubrovalvata polysaccharide(CDRP)on alcoholic liver injury in rats,so as to provide theoretical support for the application and resource utilization of CDRP product.Methods:1.Preparation,content determination,monosaccharide composition,Fourier transform infrared spectroscopy analysis and nuclear magnetic resonance analysis of crude Dictyophora rubrovalvata polysaccharide: the content of crude polysaccharide was determined by phenol sulphuric acid method.The monosaccharide composition was analyzed by PMP pre-column derivatization combined with high performance liquid chromatography(HPLC).Its structure was preliminarily analyzed by FT-IR and NMR.2.In vitro antioxidant experiment of crude Dictyophora rubrovalvata polysaccharide: DPPH radical,ferric ion reducing antioxidant ability and hydroxyl radical scavenging assay were used to determine its antioxidant capacity in vitro.3.Experiment on the protective effect of crude Dictyophora rubrovalvata polysaccharide on alcoholic liver injury in rats.60 male SD rats were randomly divided into 6 groups according to body weight: normal control(NC),model control(MC),positive control(PC),low-dose(LCDRP),middle-dose(MCDRP),and highdose crude Dictyophora rubrovalvata polysaccharides(HCDRP)groups.After continuous intragastric administration for 28 days,all rats were euthanized.(1)The levels of AST,ALT and TG in serum were measured by automatic blood biochemistry analyzer,and the degree of protection of CDRP against alcoholic liver injury in rats was analyzed according to pathological sections;(2)The levels of hepatic SOD,GSH and MDA were measured by spectrophotometric method;the levels of hepatic TNF-α and IL-6 were measured by Elisa method;(3)Real-time PCR was used to detect the m RNA expression levels of Nrf2,HO-1,NQO1,GCLM,SOD1 and the inflammatory factors TLR4,NF-κB p65,TNF-α and IL-1β;(4)The protein expression levels of Nrf2,HO-1,TLR4 and NF-κB p65 were assayed by Western Blot,and the effect of crude Dictyophora rubrovalvata polysaccharide on the antioxidant capacity and anti-inflammatory capacity of alcoholic liver injury in rats was determined.Results:1.After deproteinization by Sevage method,the content of dried crude polysaccharide was 74.68%±1.32% measured by phenol-sulfuric acid method.The monosaccharide composition of polysaccharide was determined as mannose:glucuronic acid: glucose: galactose=5.06:0.67:31.5:1.33 by PMP precolumn derivations combined with HPLC.Preliminary analysis by Fourier transform infrared spectroscopy(FT-IR)and NMR showed that the polysaccharide was pyranoid ring containing α-glycosidic bond and β-glycosidic bond.2.The crude Dictyophora rubrovalvata polysaccharide has strong antioxidant ability in vitro: when the crude Dictyophora rubrovalvata polysaccharide concentration reached 3.0mg/m L,DPPH radical scavenging rate reached 80.12%,its ferric ion reducing antioxidant ability was 0.31,and hydroxyl radical scavenging rate reached 88.07%.3.Protective effect of crude Dictyophora rubrovalvata polysaccharide on alcoholic liver injury in rats:(1)The levels of AST,ALT and TG in serum of rats were significantly increased by alcohol,and were significantly decreased after the intervention of crude Dictyophora rubrovalvata polysaccharide(P<0.05).The liver morphology of the rats was observed by pathological section.The results showed that the liver cells in the NC group had regular morphology,orderly arrangement of liver cords,had no rupture of cell boundaries,and no necrosis and inflammatory cell infiltration.MC group showed severe injury: large inflammatory cell infiltration,liver cell necrosis and so on.The cell morphology of PC group tended to be normal.With the increase of polysaccharide dose,the number of cell necrosis decreased gradually,with only a few vacuoles and inflammatory infiltration,and there was a dose effect.(2)Crude Dictyophora rubrovalvata polysaccharide increased the antioxidant capacity of liver in rats with alcoholic liver injury: alcohol decreased the contents of SOD and GSH,increased the content of MDA,and down-regulated the m RNA and protein expression levels of antioxidant related enzymes.The SOD and GSH contents were increased and MDA contents were decreased after the crude Dictyophora rubrovalvata polysaccharide intervention.The m RNA expression levels of Nrf2,HO-1,NQO1,GCLM and SOD1 and the protein expression levels of Nrf2 and HO-1 were up-regulated in PC group and middle-high dose crude Dictyophora rubrovalvata polysaccharide groups(P<0.05),which alleviated alcoholic liver injury by activating Nrf2/HO-1 signaling pathway.(3)Crude Dictyophora rubrovalvata polysaccharide can reduce alcohol-induced liver inflammation in rats: alcohol significantly increased the levels of TNF-α and IL-6 in liver,and up-regulated the m RNA and protein expressions of inflammatory factors.The levels of TNF-α and IL-6 were decreased after the intervention of crude Dictyophora rubrovalvata polysaccharide.The m RNA expression levels of TLR4,NF-κB p65,TNF-α and IL-1β and the protein expression levels of TLR4 and NF-κB p65 were down-regulated in PC group and middle-high dose crude Dictyophora rubrovalvata polysaccharide groups(P<0.05),which alleviated alcoholic liver injury by inhibiting TLR4/NF-κB p65 signaling pathway.Conclusions:After deproteinization by Sevage method,the content of dried crude Dictyophora rubrovalvata polysaccharide was 74.68%±1.32% by phenol-sulfuric acid method.Its monosaccharide composition was mannose: glucuronic acid: glucose:galactose=5.06:0.67:31.5:1.33.It was a pyran ring polysaccharide containing α-glycosidic bond and β-glycosidic bond.Crude Dictyophora rubrovalvata polysaccharide has strong scavenging effect on DPPH free radical and hydroxyl free radical and strong ferric ion reducing antioxidant ability,which indicated that it had certain antioxidant ability in vitro.The results of animal experiments showed that crude Dictyophora rubrovalvata polysaccharide could alleviate alcohol-induced liver injury in rats,and the alleviating effects were related to enhanced antioxidant capacity and decreased expression of inflammatory factors.The mechanism might be related to activation of Nrf2/HO-1 signaling pathway and inhibition of TLR4/NF-κB signaling pathway.Crude Dictyophora rubrovalvata polysaccharide can be used as a functional factor to alleviate alcoholic liver injury.