Structural Characterization And Chronic Hepatic-injury Repair Of Mycelia Polysaccharides From Oudemansiella Radicata

Polysaccharides from Oudemansiella radicata have anti-inflammatory,antioxidant,antitumor and so on.However,its protective effect and mechanism on carbon tetrachloride?CCl₄?-induced chronic liver injury of mice have been little reported.In our present work,single factor test and response surface methodology?RSM?were used to determine the optimal extraction condition of mycelia polysaccharides of O.radicata?MPO?,and MPO-W was obtained by isolation and purification.Its preliminary characterization and hepatic injury-repair mechanism in carbon tetrachloride?CCl₄?-induced chronic liver injury mice were explored preliminarily.The result provided a theoretical basis for finding and discovering natural hepatoprotective drugs.The main findings were as follows:?1?The optimal extraction condition of MPO was extraction time 3.37h,water volume27.93mL,temperature 90^oC and pH 6.96,the MPO theory yield reached 1.64%.For convience for extraction,when the condition was extraction time 3.5h,water volume 30mL,temperature 90^oC and pH 7,the theory yield of MPO was 1.62%and the actual yield was1.67±0.14%.?2?After depigmentation and deproteinization,MPO was further separated and purified by DEAE-52 cellulose anion-exchange column chromatography and Sephadex G-100 gel permeation chromatography.In result,the main fraction?MPO-W?with the purity of91.73±2.63%was acquired.?3?Physicochemical properties showed that MPO-W,a light earthy yellow powdery substance,was insoluble in most organic solvents,had good thermal stability,a degree of swelling of 134.81±3.93%,water retention capacity of 4.55±0.29%and oil holding capacity of 2.38±0.05%.Gas chromatography-mass spectrometry?GC-MS?and high-performance gel-permeation chromatography?HPGPC?displayed that MPO-W with molecular weight of1.30×10⁵Da was made up of fucose,mannose,galactose and glucose with a molar ratio of8.67:37.89:35.98:16.60.Based on the analysis results of Fourier transform-infrared spectroscopy?FT-IR?,nuclear magnetic resonance?NMR?and methylation,MPO-W was a pyranose with?-and?-configurations containing six monosaccharide sugar residues including Manp-?1?,?3?-Fucp-?1?,?2?-Manp-?1?,?4?-Glcp-?1?,?6?-Galp-?1?and?2,6?-Manp-(1?.Scanning electron microscopy and Congo red test results revealed that MPO-W with a large intramolecular force and a compact structure did not have a three-dimensional spiral space conformation.?4?In vitro antioxidant capacity of MPO-W was evaluated by measuring scavenging effects on hydroxyl,superoxide anion and 1,1-diphenyl-2-picrylhydrazyl?DPPH?free radicals,as well as reducing power.The results manifested that MPO-W had some capacity of scavenging free radicals and reducing power.?5?Hepatic injury-repair abilities of MPO-W in CCl₄-induced chronic liver injury mice were investigated.?1?For metabolizing enzyme of CCl₄,MPO-W could well improve the activity and mRNA expression of cytochrome P4502E1?CYP2E1?.?2?Regarding the degree of liver damage,the activities of marker enzymes in the serum of chronic liver injury mice were significantly inhibited by MPO-W,and histopathological sections of mice liver also showed that MPO-W could reduce liver damage.?3?For the metabolism disorders of liver and blood lipid,MPO-W could effectively regulate hepatic adenosine monophosphate activated protein kinase-??AMPK??and sterol regulatory element binding proteins-1c?SREBP-1c?in chronic liver injury mice to reduce the accumulation of TC and TG,and also improve the dyslipidemia.?4?For in vivo antioxidant capacity,MPO-W significantly enhanced the activities of antioxidant enzymes and total antioxidant capability?T-AOC?,as well as reduced malondialdehyde?MDA?and lipid peroxide?LPO?contents in liver,and the analysis results of antioxidant-related proteins and fluorescent probe?DHE?-labeled ROS showed that MPO-W distinctly up-regulated the protein and mRNA expressions of heme oxygenase-1?HO-1?and red-derived nuclear factor erythroid 2-related factor 2?Nrf2?,and decreased ROS levels in hepatocytes,further proving that MPO-W had good antioxidant activity.?5?For anti-fibrosis ability,MPO-W apparently down-regulated the protein and mRNA expressions of transforming growth factor?1?TGF?1?,thereby inhibiting the activation of hepatic stellate cells,and the analysis results of Masson,Sirius red staining and hydroxyproline?Hyp?also supported the anti-fibrotic effect of MPO-W.?6?For inflammatory response,MPO-W effectively suppressed the phosphorylation levels of I?B kinase?IKK?,nuclear factor-?B P65?NF-?B P65?and the degradation of inhibitor of NF-?B-??I?B??to prevent the activation of NF-?B for reducing the secretion of inflammatory factors,thereby achieving the purpose of anti-inflammatory.?7?For apoptosis,MPO-W significantly evaluated B-cell lymphoma-2?Bcl-2?,down-regulated cysteinyl aspartate specific proteinase-3?Caspase-3?,and the number of fluorescent-labeled apoptotic cells by Tunel also indicated that MPO-W had anti-apoptosis ability.?8?For autophagy,the protein expression of autophagy-related protein-5?ATG5?,ATG7,and microtubule associated protein 1A/1B-light chain 3-II?LC3-II?/LC3-I ratio was obviously decreased by MPO-W,testifying that MPO-W had the ability to relieve autophagy.The in vivo test results showed that MPO-W had the molecular regulating mechanisms of antioxidant,fibrosis,inflammation,apoptosis and autophagy against CCl₄-induced chronic liver injury.