The Effect And Mechanism Of Polysaccharides From The Barley On The Colorectal Cancer HT-29 Cells

Barley has a long history ofplanting in the history of China,the source of our country is quite common.Barley polysaccharides are a kind of bioactive substance.While the studies on biological activity of barley in China are relatively few at present.In this article,the barley was used as raw material in order to screen a kind of barley which is better in anti-tumor.And explore its molecular mechanisms which can provide the corresponding theoretical basis for the comprehensive utilization of barley.In this experiment,MTT was used to screen the barley with better inhibitory effect on HT-29 cells from 13 kinds of barleys.The results showed that the 25th barley was better effective on HT-29 cells whose inhibition rate reached 40%.Extracted barley polysaccharides from 25th barley by water-extraction and alcohol-precpitation method.Three alcohol components had been gained,they were 50%,60%and 70%alcohol components respectively.MTT was used to detect the inhibition effect on HT-29 cells of different alcohol components.The results indicated that the inhibitory effect of 50%alcohol components were better than others.So 50%alcohol components were selected for the following experiments.After degreasing,deproteinization and other treatments,50%alcohol components were separated and purified by CL-4B Sepharose chromatography(id1.6×60 cm).Five components had been acquired(BP-1,BP-2,BP-3,BP-4 and BP-5).MTT was used to detect the activity of different components.As a result,BP-1 component was better than others.BP-1 was a single-component of 6776 kDa which was confirmed by HPLC,with 96.83%of the average total sugar content and nonprotein.Instrumental analysis methods(Color test,UV,IR,GC)were used to characterize the structure of BP-1,showed that BP-1 was P-D-pyranose polysaccharide without reduced carbonyl,phenolic hydroxyl,free protein and nucleic acid substances,which composed of rhamnose,arabinose,xylose,glucose with molar ratio of 1.00:1.50:1.92:8.82.The MTT test indicated that BP-1 was better effective on HT-29 cells,and presented the concentration and time dependence.The half inhibition concentration on HT-29 cells was about 48.18 g/mL at the time of 48h.The results of DAPI staining,AO/EB staining,Scanning Electron Microscope and Flow cytometric analysis(FCM)indicated that BP-1 inducing HT-29 cells apoptosis based on the typical apoptotic morphological changes,apoptosis of HT-29 cells were caused through cells arrested at G0/G1 and the apoptotic rates significantly increased with prolonging the cultured time.The membrane potential of mitochondria was significantly decreased by Luo Danming staining.The results of Fluorescence staining and Western blot showed that the membrane potential of mitochondria was decreased and generation of intracellular reactive oxygen species(ROS)was increased in HT-29 cells after treated with BP-1.The large number of ROS produced in the cells caused the sharp decrease of Ras expression,which led to the decrease of p-ERK expression and up-regulation of p-JNK expression respectively.Moreover prevented the degradation of IκB-α and suppressed NF-κB p65 to enter the nucleus.Thus influenced the efficiency of Bcl-2.The ratio of Bax/Bcl-2 was increased,which led to the release of Cytochrome c to cytoplasm from mitochondria,then followed a series of Caspases cascade reaction,and eventually induced the apoptosis of HT-29 cells.