Alleviation And The Protective Mechanism Of Selenium Polysaccharides From Camellia Oleifera Fruit Shell On The Manganese-induced Damage

Camellia oleifera is known as the world’s four largest woody oil plants with Canarium oleosum,Elaeis guineensis and Cocos nucifera.C.oleifera fruit shell is one of the by-products in the camellia industry with a huge output lack of utilization.Manganese is a necessary trace element in the human body,but too much manganese intake can cause manganese poisoning.In recent years,the concentration of manganese in the environment has gradually increased,resulting in the increase in risk of manganese poisoning of ordinary people,but the effective protection and treatment measures for manganese poisoning have yet to be studied.Therefore,in order to improve the utilization rate of shell and provide a reference for the prevention and control of manganese poisoning,this study uses the C.oleifera fruit shell as the research material and extract polysaccharides with hot water extraction.After purification,selenium modification of sodium nitrate-sodium selenate was applied to obtain selenium polysaccharides and its physical and chemical properties were then analyzed.Hep G2 cells were set as a model to evaluate cell viability,antioxidant enzymes,and active oxygen levels,etc.,to explore the relief and mechanism of selenium polysaccharides on manganese-induced cell.Then Caenorhabditis elegans was applied to assess the role and mechanism of selenium polysaccharides to alleviate manganese damage in vivo.Below are several research findings:1.The purified camellia polysaccharides（PCS）through the hot water extraction and purification,is selenium modified to obtain selenium polysaccharides（Se-PCS）as the content of Se is 444.9±1.02μg/g.The polysaccharide content of Se-PCS is 86.15±0.37%,the protein content is 1.01±0.04%,and the molecular weight is 62.23 k Da.Se-PCS is composed of xylose,mannose,glucose,and lactose as the molar ratio is 45.17:5.27:36.03:13.53.PCS’s selenium modification does not affect the main structure of polysaccharides.Compared with PCS,Se-PCS not only has typical polysaccharide structure characteristics,but also the new selenium polysaccharide characteristic structure is also formed in the Se-PCS after selenium,in which 761.83 cm^-1 and 669.25 cm^-1 have an absorption peak caused by Se=O and Se-O-C.2.The Se-PCS with a concentration of 0.1-1.6 mg/m L increases the survival rate of cells under manganese stress,up to 76.86%and 76.60%.And the antioxidant enzymes of cells were obviously promoted after Se-PCS treatment,activating oxidation defense system.Then the ROS levels and MDA content were reduced by 55.58%and 62.76%,respectively.Moreover,the vitalities of Caspase-9 and Caspase-3 were decreased,mitochondria membrane potential was increased,and the number of apoptosis cells is reduced.These show that Se-PCS has a strong relief effect on Hep G2 cell damage induced by manganese.3.Under the manganese stress,Se-PCS（0.3 mg/m L）can extend the survival time of C.elegans by 33.60%,and improve the antioxidant enzyme activity,then reduce the ROS and MDA content of the nematode by 55.99%and 52.23%,respectively.4.Further analysis found that Se-PCS can regulate the related gene expression of the insulin signal pathway（IIS pathway）in C.elegans.Under manganese stress,Se-PCS can reduce the expression of daf-2,akt-2,pdk-1 and age-1 genes in the IIS pathway,meaning the IIS pathway suppressed and the expressions of the daf-16 and skn-1 gene were activated.Se-PCS also makes the DAF-16 transcription factor into the nuclear.Furthermore,Se-PCS fails to prolong the survival time of different mutants involving in daf-2,akt-2,pdk-1,age-1,daf-16 and skn-1 genes under manganese stress.However,the expression of the sgk-1 gene is not significantly changed and Se-PCS can extend survival time of sgk-1 mutant,indicating sgk-1 does not participate in the regulation of the Se-PCS.Meanwhile,Se-PCS treatment can also activate the expression of hsf-1,sod-3,ctl-2,gst-1and gst-4 genes in oxidation defense system,which is in the downstream of daf-16 gene.These results show that the Se-PCS mainly regulates the gene expression of the IIS pathway and activates daf-16 gene,thereby activating the oxidation defense system to alleviate the manganese-induced damage.