| Flammulina velutipes is one of large edible fungus,containing a variety of bioactive substances.Polysaccharide is one of the most important active substances in Flammulina velutipes.The biological activities of FVP(Flammulina velutipes polysaccharide)were widely studied and reported.But there are fewer reports on the anti-tumor effect of FVP,and the mechanism is not clear.In the early stage of the laboratory,Flammulina velutipes polysaccharides were isolated from the fruiting bodies of Flammulina velutipes,and the effect of Flammulina velutipes polysaccharides on proliferation Hep G2 cells was preliminarily detected.Therefore,this study further investigates the effect of Flammulina velutipes polysaccharide on proliferation and apoptosis of Hepatocellular Carcinoma(Hep G2)cell and its potential mechanism,and provides the experimental basis for the anti-tumor effect theory and mechanism of Flammulina velutipes polysaccharide.Methods: MTT assay was used to detect the inhibitory effects of four polysaccharides including 45%,75% alcohol precipitation components of Flammulina velutipes polysaccharides and its corresponding in addition to protein polysaccharides FVP1、FVP2、FVP1a、FVP2a on the proliferation of Hep G2 cells at different concentrations(0 μg/m L,50 μg/m L,100 μg/m L,200 μg/m L,500 μg/m L,1000 μg/m L)and different time(24 h、48h)respectively.As a basis,FVP1 with obvious inhibitory effect and concentrations(0 μg/m L,50 μg/m L,100 μg/m L,200 μg/m L,500 μg/m L,1000 μg/m L)at different time(24 h、48h)were selected for the following experimental conditions.The effect of FVP on the apoptosis rate of Hep G2 cells was detected by Annexin V-FITC/PI double staining kit.The morphological changes of apoptosis after treatment with FVP1 were detected by Hoechst33258 fluorescent staining.The expressions of Caspase-3,8,9,Bax and Bcl2 gene m RNA expression level were detected by q PCR(real-time fluorescence quantitative PCR).The activation of apoptotic protein Caspase-3,8,9 was detected by kit method.The protein expressions level of Bcl2,Bax and PARP were detected by western blot.Results:1.The results of MTT showed that four polysaccharides had inhibitory effects on the proliferation of Hep G2 in the experimental concentration range and different time 24,48 h,with concentration-dependent and time-dependent relationgship.The inhibitory effect was FVP1>FVP1a>FVP2>FVP2a,that is,75% of the component inhibition effect was better than 45% of the component effect,and no treated polysaccharide was better than no protein polysaccharide2.The results of Annexin V-FITC / PI double staining showed that the apoptotic rate(p<0.05)was increased after FVP1 treated for 24 h and 48 h.With the concentration increased rate of apoptosis increased,and the apoptosis rate at 48 h was higher than at 24 h.3.Hoechst 33258 fluorescence staining showed that with the increase of FVP1 concentration,the chromatin of nucleus showed typical changes of dense and bright blue stained cells,and the number of cells observed decreased with the increase of FVP1 concentration.4.The results of q PCR showed that FVP1 could significantly increase the gene m RNA expression level of Caspase-9,Bax(p<0.05).The effect of 48 h was better than that of 24 h.FVP1 could increase the gene m RNA expression level of Caspase-3,8,but the effect was not significant.And FVP1 had no significant effect on the gene m RNA expression level of Bcl 2.It’s suggested that FVP1 may induce Hep G2 cell apoptosis by affecting mitochondrial apoptosis and death receptor pathway.5.Caspase protein activity experiments showed that FVP1 could significantly increase the activity of Caspase-3,8 and 9(p<0.05),and further confirm that Caspase-3、8、9 protein played a role effect in the process of apoptosis.6.The results of western blot showed that FVP1 could significantly increase the expression of pro-apoptotic protein Bax,inhibit the expression of anti-apoptotic protein Bcl2 and significantly promote the PARP cleavage activation level.It was suggested that FVP1 induced apoptosis by influencing the expression of key protein Bax Bcl2 in mitochondrial apoptotic pathway.In summary,FVP1 can inhibit the proliferation and induce apoptosis of Hep G2 cell,by promoting the expression level of pro-apoptotic protein Bax and m RNA level;inhibiting the expression of anti-apoptotic protein Bcl2 protein affect mitochondrial permeability and increasing Caspase-9,3 protein,then activating the apoptotic substrate PARP.At the same time,the activity of Caspase-8 protein was increased,indicating that the death receptor pathway was also involved in the process of apoptosis induced by FVP1.It was proved that FVP1 can inhibit the proliferation and induce apoptosis of Hep G2 in vitro.Deeply specific mechanisms need further study. |
