Based on previous report that the Chinese herb Ligustrum lucidum (LL) extract directly inhibited hepatitis C virus (HCV) replicase (NS5B) activity, the active components of LL extract to inhibit HCV NS5B activity and their inhibition mode were investigated in this study. LL extract was separated using ethyl acetate and thin layer chromatography (TLC). The inhibitory activity of separated fractions on HCV NS5B was analyzed by the inhibitory assay of NS5B activity. The results showed that only fractions 1 and 2 inhibited NS5B activity, and fraction 2 possessed higher inhibitory activity than fraction 1. HPLC analysis combined with inhibitory assays indicated that ursolic acid and oleanolic acid are the active components within fractions 1 and 2 to inhibit NS5B activity, separately. Moreover, oleanolic acid possessed higher inhibitory activity than ursolic acid. Further inhibition mode analysis found that both oleanolic acid and ursolic acid suppressed NS5B activity as noncompetitive inhibitors. The Ki values of ursolic acid and oleanolic acid were about 4.7 microg x mL(-1) (10 micromol x kg(-1)) and 2.5 microg x mL(-1) (5.5 micromol x kg(-1)), respectively. Taken together, these results demonstrated that oleanolic acid and ursolic acid suppressed NS5B activity as noncompetitive inhibitors, implying that the two natural products have potential value for HCV therapy.

Objective To investigate the effects of growth differentiation factor 11 (GDF11) on proliferation of mouse neural stem cells (NSCs) and expression levels of transforming growth factor (TGF)-β/Smads and Wnt/β-Catenin signal key proteins.Methods NSCs,derived from the subventricular zone of E14 d CD1 mice,were cultured and induced differentiation;specific proteins nestin and SOX2 were confirmed by immunofluorescence assay.Neuron marker nucleus antigen (NeuN)and astrocyte marker glial fibrillary acidic protein (GFAP) were identified by immunofluorescent staining.The cells of third generation in their exponential phase were chosen and randomly divided into experimental group (adding GDF11 to make the final concentration as 40 ng/mL) and control group (adding equal amount of culture fluid).The proliferation of the cells in the two groups was detected by 5-ethynyl-2'-deoxyuridine (EdU) kits and protein expressions of Smad2/3,phosphorylated (p)-Smad2/3,Smad4 and β-Catenin were measured by Western blotting one and 6 h after treatment.Results Round and bright cells suspended in culture medium were observed through optical microscope.Immunofluorescence assay showed that over 90％ cells expressed both nestin and SOX2,and some of them expressed NeuN or GFAP.EdU proliferation test showed that the percentage of EdU positive cells in the experimental group (0.34±0.08) was significantly higher than that in the control group (0.24±0.03,P＜0.05).Western blotting showed that the expression levels ofp-Smad2/3,Smad4 and β-Catenin were significantly increased one and 6 h after treatment as compared with those in the control group (P＜0.05).Conclusion GDF11 can promote the proliferation of NSCs in vitro and probably is on account of activating TGF-β/Smads and Wnt/β-Catenin signal pathways.

Objective To investigate the effect ofmiR-184 on proliferation of neural stem cells (NSCs) and its mechanisms in mice.Methods The pHBLV-U6-GFP-miR-184 over-expression plasmid and pHBLV-U6-GFP-miR-184 inhibitor plasmid were used to construct recombinant lentivirus.And the NSCs derived fiom subventricular zone of E14d CD1 mouse were confirmed by immunofluorescence assay.There were four groups that contain a miR-184 overexpression group,a miR-184 inhibitor group and two control groups.The NSCs which infected with lentiviral vectors were selected for puromycin resistance for 5-7 days,and then surviving cells were cultivated to three generations.The expression level ofmiR-184 was detected by real time-quantitative PCR (RT-qPCR).And the target genes ofmiR-184 were predicted through TargetScan,IRTarBase and MiRanda,and were confirmed by Western blotting and RT-qPCR.The cells in the four groups were culttared under proliferating conditions incorporated bromodeoxyuridine (BrdU) in cell proliferation analyses.The protein expressions of Hesl and Hes5,the target proteins of Notch signaling pathways,and their mRNA expressions were detected by Western blotting and RT-qPCR.Results There were 90％ of cells in each group expressing both Nestin and Sox2.The miR-184 level in the miR-184 overexpression group was 67.63±7.53 times of that of the control group,with significant difference (P＜0.05).The percent of BrdU+/DAPI+ of the miR-184 overexpression group was 1.47±0.05 times of that in the control group,with significant difference (P＜0.05);and the percent of BrdU+/DAPI+ of the miR-184 inhibitor group was 0.84±0.03 times of that in the inhibitor control group,with significant difference (P＜0.05).Numbl was a target gene ofmiR-184 indicated by IRTarBase and MiRanda.The miR-184 could inhibit Numbl protein expression;the Numbl protein expression level in the miR-184 overexpression group was 0.73±0.07times of that in the control group,and the Numbl protein expression level in the miR-184 inhibitor group was 1.30±0.05 times of that in the control group,with significant difference (P＜0.05);but miR-184 did not change the Numbl mRNA level.The miR-184 could activate Notch signaling pathway through inhibiting the Numbl protein expression,and increase the Hes1 and Hes5 protein and mRNA expression levels (P＜0.05).Conclusion The miR-184 promotes the NSCs proliferation through inhibiting the Numbl protein translation and further activating the Notch signaling pathway.

Objective To explore the effect of R-spondin3 on the proliferation of neural stem cells (NSCs) and its mechanism in mice.Methods The mouse NSCs derived from the subventricular zone of E 14-15d CD 1 mice were confirmed by immunofluorescence assay.The NSCs after 3 passages of culture were chosen and randomly divided into 2 groups (V=1 mL).In the experimental group,0.8 μL of R-spondin3 with an initial concentration of 50 μg/mL was added (final concentration:40 ng/mL) while in the control group an equal amount of culture fluid was added.The proliferation of the cells in the 2 groups was detected by 5-Bromo-2-deoxy Uridine (BrdU) kits after the cells were treated by R-spondin3 for 6 hours.The protein expression of [β-catenin was measured by western blotting after the cells were treated by R-spondin3 for 4 and 8 hours.Results Under optical microscopy,the round and bright cells grew in culture medium and easily accumulated to become neurospheres.Immunofluorescence assay showed that over 90％ of the cells expressed Nestin and SOX2 and that some of them expressed NeuN or GFAP after induced differentiation.Brdu proliferation test showed that the proliferation rate of Brdu+/DAPI+ for the experimental group (1.56±0.03) was significantly higher than that for the control group (1.04±0.04) (P＜0.05).Western blotting showed that the expression levels of [β-catenin were increased at both 4h and 8h after treatment for the experimental group (1.09±0.10 and 1.20±0.13),significantly higher than those for the control group (0.56±0.05 and 0.83±0.04) (P＜0.05).Conclusions R-spondin3 can promote in vitro proliferation of NSCs in mice,which may be associated with activated Wnt/β-catenin signal pathways.

Objective To investigate the effect of galangin on proliferation and apoptosis of glioma cells in vitro.Methods (1) The glioma cells U87 and U25 1were divided into blank control group,DMSO group,100,200,300 and 400 μmol/L galangin treatment groups.MTT was used to study the effects of drugs on the proliferation of U251 and U87 cells.(2) Hoechest staining was used to observe cell apoptosis in the presence of different concentrations of galangin (0,100 and 200 μmol/L).(3) Flow cytometry was employed to detect the apoptosis of U251 and U87 cells in the presence of different concentrations of galangin (1 00 and 200 μmol/L).(4) Western blotting was employed to detect the expressions of apoptosis-related protein 3-Catenin,B-cell lymphoma-2 (Bcl-2),Bcl-2 related protein gene (Bax),cleaved-caspase-3,cleaved-caspase-9 and poly (ADP-ribose) polymerase (PARP) in the presence of different concentrations of galangin.Results (1) The proliferation of U251 and U87 cells was obviously inhibited atter 100,200,300 and 400 μmol/L galangin treatments,and dose-effect relation was noted.The concentrations of galangin at half rate of inhibition (IC50) were 281,321,276 and 229 μmol/L in U251 cells,and 289.4,261.1,247.4 and 225.3 μ mol/L in the U87 cells after 100,200,300 and 400μmol/L galangin treatments for 24 h.(2) Under the action of galangin,corresponding increase in apoptosis rates of U251 and U87 cells was noted following the increase of galangin concentrations (0,100 and 200 μmol/L),with significant differences (P＜0.05).(3) The detection of cell apoptosis by flow cytometry found similar changes.(4) Western blotting results indicated that galangin at the concentration of 0,100 and 200 μmol/L could significantly decrease the expressions of apoptosis-related protein 3-Catenin and Bcl-2,and increase the Bax,cleaved-caspase-3 and cleaved-caspase-9,and cleaved-PARP expressions;significant differences were noted between each two concentrations (P＜0.05).Conclusion Galangin can inhibit proliferation of glioma cells U251 and U87,and induce mitochondrial pathway of apoptosis via Wnt/β-Catenin signaling.