Effect Of Cibotium Barometz Polysaccharides On Proliferation And Oxyzen Radical In Cnondrocytes

ObjectiveTo evaluate the effects of Cibotium barometz polysaccharides(CBPS) on proliferation and oxygen radical of chondrocytes cultured in vitro so as to provide some evidences for clinical application of CBPS.Methods1.The CBPS were extracted by hot water, alcohol precipitation and protein filtration with Sevag reagent; The content of the polysaccharides was determined by phenol-sulfuric acid colorimetry.2.Establishment of chondrocytes system cultured in vitro isolated from knee articular cartilage of 4-week old SD rats; Morphological changes were observed by inverted phase contrast microscope; The effect of proliferation and the evaluation for the phase of cell cycle treated by CBPS were dected by MTT assay and flow cytometry respectively; The expression of related mRNA and protein on proliferation were tested by RT-PCR and Western Blot while the OD value of SOD^ MDA and NO were assessed by enzyme-linked immuno sorbent assay.Results1.The content of polysaccharides was 76.91% with good precision of RSD at 1.86% as well as fine stability of RSD at 1.98%, in addition, the result of average recovery test was 95.4% with the RSD at 1.83%, all showed that the CBPS was at a optimized level.2.The characteristics of the passage 2 chondrocytes were presented:the almost coincident shapes and moderate counts, with clear boundaries and distinct nuclei; The nuclei was watched in blue stained by toluidine blue; The cytoplasm of chondrocytes was identified in brownish yellow stained by immunohistochemical staining in positive group.3.MTT assay analysis revealed that the administration of CBPS activated the proliferation of chondrocytes as evidenced in a certain dose-dependent and time-dependent manner associated with the best time and concentration at 48 h and 200μg/ml respectively; Compared with the normal group, the CBPS treated groups(100;200;400 μg/ml) caused a marked decrease of the percentage proportion in the G0/G1 phase while increase in the S phase detected by Flow Cytometer; RT-PCR and Western Blot suggested that CBPS upregulation of the expression of mRNA and protein in CDK.4, Cyclin D1 and pRB as compared to the normal group, especially given significant difference from the 200 μg/ml group(P<0.01).4.Significant differences with the expression of SOD、 NO、MDA in model groups were markedly presented as evidenced the establishment of the model required compared with the normal group by enzyme-linked immuno sorbent assay(P<0.01); Compared with the model group, as demonstrated by elevation of the expression of SOD while decline of the expression of NO and MDA, CBPS(100;200;400;800 μg/ml) can significantly protect against the oxidative stress as indicated especially given significant difference from the 200 μg/ml group (P<0.01).Conclusions1.The proliferation of chondrocytes is promoted by CBPS in a certain dose-dependent and time-dependent manner with the underlying mechanisma by improving transformation rate in the cell cycle G1 to S phase and upregulate the mRNA and protein expression of positive factors.2.CBPS alleviated the expression of abnormal oxygen radical while increased the level of antioxidative factor which exerted the function of antioxidation.