The Research On The Effects Of Total Flzvonoids Of Broussonetia Papyrifera On Proliferation And Apoptosis Of Hepatocellular Carcinoma Cell HepG-2

Objective:1Total flavonoids was extracted from broussonetia papyrifera by microwave-assisted extraction. The extractives were purified with macroporous resin and polyamide column, which used for primarily identified.2To observe the influence of total flzvonoids of broussonetia papyrifera on inhibiting the growth of hepatocellular carcinoma cell line HepG-2, and determine the quantities-effect, choose inhibiting concentration (IC50) for the reference of the further studies on anti-hepatoma by TFBP.3To provide scientific experimental basis and reliable methods for development and utilization of broussonetia papyrifera resources.Methods:1Total flavonoids was extracted from broussonetia papyrifera by microwave-assisted extraction and then the extractives were isolated and purified.2Determining the inhibitive rate of HepG-2growth by different concentrations of TFBP after24h,48h and72h with MTT method.3Cell morphological change was observed by inverted microscope when HepG-2cells were induced by different concentrations of TFBP.4Observing the apoptosis morphological change when HepG-2cells were induced by different concentrations of TFBP after48h by fluorescent staining with Hoechst33342staining.5The protein expression of Bcl-2、Bax was detected using immunohistochemical assay when HepG-2cells were induced apoptosis by TFBP.6The expression changes of Caspase-3、Bcl-2、Bax was detected using RT-PCR before and after induced by TFBP.Results:1The extraction rate was139mg/100g after extracted by microwave-assisted and purified.2MTT results showed that the growth inhibitory effect of HepG-2cells induced by TFBP was time-dependent and dose-dependent. In experiment group,3,6mg/ml of TFBP on inhibition of HepG-2cells was not obvious after24hours(P>0.05), but the result of9,12mg/ml had a high degree of statistical significance on inhibition of HepG-2cells compared with the control group(P<0.05).3,6,9mg/ml of TFBP all had inhibit effect on HepG-2cells after48hours and72hours. It inhibited the proliferation of HepG-2cells in a time and dose dependent manner as the concentrations of TFBP increased gradually. The different concentration of inhibit rate was37%,54%,63%respectively after48hours.3,6,9mg/ml were chosen as the experimental concentration while6mg/ml was used for observing the apoptosis morphological.3Under an inverted microscope it can be seen that in the control group HepG-2cells were in spindle shape, morphology of ceils was intact, diffusely arranged into patches, abundant cytoplasm and uniform distribution of large nuclei; After induced by6mg/ml of TFBP for48h, it can be observed that cell derangement, irregular contour, membrane shrinkage, change the park, off the uneven distribution of cytoplasm within the cell, the cell size becomes smaller and fewer number of cells. With the method of fluorescent staining with Hoechst33342staining in control group, it can be seen that HepG-2cells membrane integrity, full of nuclear morphology, nuclear quality stained uniform coloring lighter; After induced by6mg/ml of TFBP for48h, it can be observed that membrane occurred shrinkage, the nucleus was relatively small, the fluorescence enhancement was obviously, the nucleus chromatin concentration and fragmentation was round cell debris, namely apoptotic bodies, which shows the typical apoptotic morphological features.4Immunohistochemical assay showed that when HepG-2cells induced by TFBP, pro-apoptotic protein Bax is up-regulated, the number of positive cells are increased and the staining of the cytoplasm was deepened. The positive rate was36.17±0.05when the drug concentration was9mg/ml, which had a statistically significant difference compared with the control group (P<0.05). The expression of inhibitor of apoptosis gene Bcl-2reduced, which performance to reduce the number of positive cells and cytoplasm coloring lighter, almost no coloring in the nucleus. The positive rate was30.45±1.52when the drug concentration was9mg/ml, which had a statistically significant difference compared with the control group (P<0.05).5RT-PCR results showed that when HepG-2cells induced by3,6,9mg/ml of TFBP, mRNA levels of Caspase-3and Bax were increased with drug concentration increasing while Bcl-2decreased(P<0.05).Conclusions:1microwave-assisted extraction method was efficient and energy-saving.2TFBP had high inhibition to the growth of human hepatoma HepG-2cells, and inhibition was time-dependent and dose-dependent. 3TFBP can induce human hepatoma HepG-2cells, which may have an relationship with increase pro-apoptotic factor Caspase-3, Bax as well as lowered inhibition of apoptotic protein Bcl-2expression.