| ObjectiveSchisandra is a kind of traditional Chinese medicine(TCM),is derived from the magnoliaceae plant of Schisandra chinensis(Turcz.)Baill.'s dry ripe fruit,habitual Known as the "North schisandrae".But if it is derived from the magnoliaceae plant of Schisandra sphenanthera Rehd.et Wils.'s dry ripe fruit,it knowns as the "Sorth schisandrae".Schisandrae contains a lot of polysaccharide composition,polysaccharide also known as carbohydrates,the general formula for the(CH2O)n.Schisandrae polysaccharide mostly with antioxidant,anti-aging effect,can protect human kidney,liver.In the existing literature of fructus schisandrae polysaccharide antioxidant and aging research is not thorough,especially the study of its mechanism of action.Therefore,to explore the antioxidant effect of fructus schisandrae polysaccharide and it' s mechanism research has a big significance.NF-E2(Nuclear factor erythriod 2-related factor 2,Nrf2)has been provened to be one of the most closed related target of antioxidant effect,when stimulated with Oxidative stress or other stimulis in vivo or in vitro,Nrf2 and Keapl rapid dissociation in the cytoplasm and Nrf2 has been activated,it moves from outside the nucleus to inside.In this study,we used human embryonic kidney cell 293T as a model to study the antioxidant effect and the effect of cell proliferation of schisandrae chinensis polysaccharides,which provide a new ideas and direction for the prevention and treatment of Oxidative stress,aging,and also provides a theoretical basis for the development of related foods and drugs of Schisandra chinensis polysaccharides.Methods1.Cell culture conditionsAccording to the conventional method training 293T cell lines,Nutrient solution prepared according to the following ratio:Dulbecco modified Eagle medium(DMEM):FBS:Penicillin-Streptomycin Solution =(45ml:5ml:500ul),Placed in the incubator equilibrated with humidified 5%C02 in air at 37?,observe the cell growth situation daily and represented in time.2.The extraction,separation and purification of schisandra chinensis crude polysaccharides(SCP)Buy the Schisandra chinensis from the pharmacy,degrease by petroleum ether and then the crude polysaccharides was obtained by water,extraction.After the de-colourisation,deproteinization,separated and purified by DEAE-52?sephadexG-100 chromatography column,one purified polysaccharide fraction was got.Then freeze-dried the filtrate and preservation it in dry and cold.3.The antioxidant effect of schisandra chinensis polysaccharidesExperimental procedures were conducted according to the kit instructions.4.The regulate function on Nrf2 and its downstream factors of schisandra chinensis polysaccharidesWestern blot were used to detect the protein expression levels of Nrf2?NQO1?HO-1 in 293T cells after administration of different concentrations of polysaccharide components.Fluorescence microplate were used to detect the activation of the ARE-luciferase reporter by different concentrations of polysaccharide components.5.The influence on the activity and stability of internal and external Nrf2 of polysaccharide componentsWestern blot were used to detect the protein expression levels of internal and external Nrf2 in 293T cells after administration of different concentrations of polysaccharide components.Western blot were used to detect the protein expression levels of Nrf2 in different time periods after administration CHX or not pretreatment of polysaccharide components for 2hr.6.The influence on Nrf2 DNA binding activity of schisandra chinensis polysaccharidesCollect and cracking of the cell after administration of polysaccharide components,collect the nucleus protein,use Electrophoretic Mobility Shift Assay Kit to detect the change of Nrf2 DNA binding activity.7.The influence of schisandra chinensis polysaccharides on cell proliferationUsing 293T cells as the research object,tertiary butyl hydroquinone(tBHQ)(10uM)used as agonists in positive control group,the experiment with different concentration of polysaccharide components on the cell,cultured for 24,48,72 h,cell proliferation status was evaluated by MTT assay.Results1.The total polysaccharide content of SCP is 40%after extracted with water,After the deproteinization,de-colourisation,with water as eluent,the polysaccharide component SCP-1 was isolated and purified by DEAE-52 column,The purified component SCP-2 was obtained after sephadexG-100 column separation and purification,total polysaccharide content of SCP-2 is 86%.2.SCP-2 can increase the expression of Nrf2 and its downstream factors,enhancement ARE luciferase activity.3.SCP-2 can increase the content of GSH and CAT in 293T cells,reduce the content of MDA,increase the vitality of SOD in 293T cells.4.SCP-2 can reduce the expression level of Nrf2 and Keapl protein in cytoplasm.activate Nrf2 and transfer it to the nucleus,meanwhile SCP-2 increase the stability of Nrf2 protein.5.SCP-2 can enhance Nrf2 DNA binding activity6.SCP-2 can promote normal cell proliferation,inhibit the proliferation of cancer cells.ConclusionSchisandra chinensis polysaccharides(SCP)can increase the expression of Nrf2 and its downstream antioxidant factors,increase the expression of endogenous antioxidant enzymes such as glycine,catalase and so on.It can also reduce the content of MDA,enhance the activity of SOD in cells.On normal cells SCP has the effect of promoting proliferation but inhibit the proliferaction of tumor cells.The mechanism of those above phenomenon is mainly because schisandra chinensis polysaccharides(SCP)promote Nrf2 movement into nuclenus,increase the Nrf2 protein stability and enhance Nrf2 DNA binding activity.It can be considered that Schisandra chinensis polysaccharides can be developed as a natural antioxidant. |
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