Structural Characterization,Anti-oxidation And Anti-inflammation Analysis Of Mycelia Selenium Polysaccharides From Hypsizygus Marmoreus SK-03

The selenium,as an essential and fundamental nutrient to organism,could participate in the physiological activities of enzymes and protect the structure and function of biomembranes against oxidative stress.The selenium deficiency may contribute to the adverse consequences for lots of disease and exacerbate the disease progression,such as inflammation and cardia-cerebrovascular diseases.The edible fungi polysaccharides,which were the important source of natural medicines,had plenty of biological activities including antioxidation,immunocompetence,anti-aging,anti-inflammation and antitumor activities.The selenium-polysaccharides were the organic combination of selenium and polysaccharides,showing higher pharmacological activities and being beneficial to absorption and utilization for human.In our work,the mycelia selenium polysaccharides?MSPS?and mycelia polysaccharides?MPS?of Hypsizygus marmoreus SK-03 were extracted,purificated and characterized.Furthermore,the anti-oxidation and anti-inflammation of MSPS in lipopolysaccharide?LPS?induced inflammatory mice were also investigated,aiming to explore mechanisms of the organic protections.The results provided new insight for natural anti-inflammation drug screening.The present results were listed as follows.?1?The optimum selenium concentrations in the process of liquid fermentation for the mycelia of H.marmoreus SK-03 were confirmed.The results showed that the optimum concentration of Na₂SeO₃ was 3mg/L.And at this concentration,the biomass yield,selenium-accumulated rate and selenium content of selenium-mycelia were 2.32±0.05g/L,21.62±1.44%and 0.28±0.03mg/g,respectively.?2?The extracted conditions of MSPS were confirmed by Plackett-Burman?PB?and response surface methodology?RSM?.Under the condition of extraction time of 3 h,ethyl alcohol concentration of 85%and ethyl alcohol multiples of 3,the MSPS yield reached the maximum of 3.19±0.35%.The purified MSPS were obtained by depigmentation,deproteinization,chromatography of DEAE-52 cellulose anion-exchange and Sephadex G-50gel permeation,dialysis,and lyophilization,and the selenium contents were 68.79±3.46?g/g.?3?The structural characterization of MSPS and MPS were analyzed.The gas chromatography?GC?analysis showed that the MSPS was composed of rhamnose,fucose,xylose,mannose,galactose and glucose with a molar ratio of 0.009:1.00:0.20:1.27:2.84:10.49,while the MPS was composed of rhamnose,fucose,ribose,xylose,mannose,galactose and glucose with a molar ratio of 0.53:3.93:0.28:0.18:2.23:12.46:6.40.The high performance liquid chromatography?HPLC?analysis indicated the the number-average molecular weights?Mn?of MSPS and MPS were 3.43×10³Da and 1.17×10³Da,respecvely.The Fourier transform infrared spectroscopy?FT-IR?analysis manifested that MSPS and MPS were pyran-type and furan-type polysaccharides,respectively,with the both configurations of?-and?-types.?4?The in vitro antioxidative activities of MSPS and MPS were measured by investigating the reducing power,and scavenging rates on hydroxide radicals and1,1-diphenyl-2-picrylhydrazyl?DPPH?radicals.The results indicated that both MSPS and MPS showed potential in vitro antioxidative activities in dose-dependent manners within the concentration range of 0³200mg/L,and the MSPS showed superior antioxidative capacities than MPS.?5?The antioxidative,anti-inflammation activities and organic protection of MSPS were analyzed in the inflammatory mice induced by intraperitoneal injection with LPS?5mg/kg?.The results showed that the activities of superoxide dismutase?SOD?,glutathione peroxidase?GSH-Px?,catalase?CAT?and total antioxidant capability?T-AOC?in lung,liver and kidney were significantly increased,while the lipid peroxidative process and the accumulation of lipid peroxidation?LPO?and malondialdehyde?MDA?were prevented by the administration of MSPS at the dose of 800mg/kg,respectively.Furthermore,after the treatment of MSPS,the serium levels of glutamyltranspeptidase?GGT?,complement 3?C3?,high-sensitivity C-reactive protein?hs-CRP?,creatinine?CRE?,blood urea nitrogen?BUN?were significantly declined when compared with that in the inflammatory mice.The levels of proinflammatory cytokines including tumor necrosis factor-??TNF-??were declined,while the anti-inflammatory cytokines levels of interleukin-10?IL-10?were increased when compared with that in the inflammatory mice.Additionally,the reactive oxygen species?ROS?levels in pneumonocyte,hepatocyte and nephrocyte could be declined after the administration of MSPS,which was measured by CM-H₂DCFDA probe fluorescence staining.Besides,the cell apoptosis could be improved by the intervations of MSPS.All the results suggested that MSPS showed superior antioxidation,anti-inflammation,and organic protections,which could be confirmed by histopathology observation.?6?The anti-inflammation mechanisms of MSPS by ROS/TLR4/NF-?B pathway were studied in lung on inflammatory mice indcued by intraperitoneal injection with LPS?5mg/kg?.After the administration of MSPS?800mg/kg?,the pulmonary wet/dry?W/D?ratio and myeloperoxidase?MPO?levels were declined comparing with that in the model mice,indicating the inflammation had been happened in lung.When compared with that in the inflammatory mice,the western blot analysis showed that the inducible nitric oxide synthase?iNOS?expressions were declined,and the immunohistochemical analysis showed that the IL-10 levels were increased after the treatment with MSPS?800mg/kg?.The real-time quantitative PCR?qRT-PCR?and western blot analysis suggested that the gene expressions and protein expressions of toll like receptor 4?TLR4?levels were declined,the I?B kinase-??IKK??activities were declined,and the phosphorylation and degradation of inhibitor of?B?I?B?were declined,resulting the inhibition on the activation of nuclear factor-?B?NF-?B?pathway,and the low expression of downstream genes.Meanwhile,the dephosphorylation of NF-?B in MSPS-administrated mice also confirmed the evidence for the inhibition on the activation of NF-?B pathway.Furthermore,the enzyme-linked immuno sorbent assay?ELISA?analysis showed that the levels of proinflammatory cytokines including TNF-?,IL-1?and IL-6 in bronchoalveolar lavage fluid?BALF?were decreased,and the pulmonary gene expression of IL-1?and protein expression of TNF-?were also declined.In summary,the results demonstrated that MSPS could prevent the inflammation by inhibiting the activation of ROS/TLR4/NF-?B pathway,playing vital roles in the organic protections.