Research On The Purification And The Mechanism Of Apoptosis On HepG2 Cells Of An Polysaccharide From Grifola Frondosa

Grifola frondosa is a widely used as medicinal fungus with good biological activity.A variety of effective anti-tumor polysaccharides have been isolated from the fruiting bodies of Grifola frondosa and mycelia in liquid culture.In this study,we used cold water to extract a new water-soluble polysaccharide from Grifola frondosa,named cGFP,and to study its effect on proliferation and apoptosis of human hepatoma HepG2 cells.The crude polysaccharide of Grifola frondosa was extracted by water extraction and alcohol precipitation at low temperature(4℃),the protein in crude polysaccharide was removed by Sevage method,the small molecule substance was removed by ultrafiltration,the macromolecular polysaccharide component cGFP,was obtained by Sephadex G-150 gel column elution.The yield of cGFP,was 1.16%.The results of high performance liquid chromatography(HPLC)showed that the molecular weight of cGFP was 1231.7 kDa.The results of ion chromatography,NMR and infrared chromatography showed that cGFP wasα-pyran polysaccharide composed of mannose,galactose and glucose with molar ratio of 1:1.48:2.05.The percentage of carbohydrate component was as high as 90.4±6.1%(w/w).The proliferation of human hepatoma HepG2 cells was significantly inhibited of 200μg/mL cGFP for 24 h by MTT assay.The apoptosis of HepG2 cells was induced in a dose-dependent manner in a certain concentration range.It was found that cGFP result in a large number of S phase cell aggregation by the detection of the PI stain.Annexin V-FITC and PI double staining showed that cGFP induced HepG2 cell apoptosis gradually from early apoptosis to late apoptosis.Scanning electron microscopy(SEM)revealed typical apoptotic morphological changes in HepG2 cells treated by cGFP.The reduction of mitochondrial membrane potential were detected by JC-1 staining and Hoechst3342 staining.Analysis of qRT-PCR showed that the expression of Bax protein increased and the expression of Bcl-2 protein decreased.The Western blot test results further show that the number of Bax proteins increased,the Bcl-2 protein content decreased,the cytochrome C(cyto-c)in the cytoplasm matrix was elevated,the caspase family protein was activated,caspase-3,and caspase-9 protein increased.It is further proved that cGFP-induced apoptosis plays a role through the mitochondrial pathway and.These results suggest that cGFP inhibits cell proliferation and induces apoptosis of HepG2 cells mainly through endogenous activation of mitochondria pathway.