OBJECTIVE To validate the anticancer effect of Angong Niuhuang pill (AGNH) and pinpoint associated molecular mechanisms using human cancer cells.METHODS Human MGC-803 gastric carcinoma and human BEL-7402 hepatocarcinoma cells were incubated with AGNH 9,30,90,300 and 900 mg·L-1 for 24,48and 72 h,respectively.Cell viability was detected with 3-(4,5-dimethylthiazol-2-yl) -5-( 3-carboxymethoxyphe-nyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and colony formation assay.Apoptosis was measured with flow cytometry and Hoechst 33258/PI staining.Change in mitochondrial membrane potential (△qψ) was detected by spectrofluorophotometer.RESULTS AGNH inhibited MGC-803 cell proliferation ( for 48 h,r =0.996,P =0.002; for72 h,r=0.756,P=0.024 ) and BEL-7402 cells (for 48 h,r =0.732,P=0.030; for72 h,r=0.702,P =0.037) in a concentration-dependent manner,as showed by MTS assay.AGNH inhibited colony formation on MGC-803 cells (r =0.914,P =0.011 ) and BEL-7402 cells (r =0.871,P =0.024) in a concentration-dependent manner for 24 h.Hoechst 33258/PI staining and flow cytometry assay showed that AGNH 900 mg·L-1 for 24 h induced apoptosis of MGC-803 and BEL-7402 cells,and the apoptosis rate was 27.2％ and 19.7％,respectively.Compared with normal control group,AGNH 900 mg·L-1 for 3 min decreased the mitochondrial membrane potential of MGC-803 and BEL-7402 cells to 15.9％ and 15.0％ of control group.CONCLUSION AGNH inhibits proliferation of human cancer cells.Apoptosis and depolarization of mitochondrial transmembrane potential are probablly its mechanism.

Though the study on ecology of Chinese traditional medicinal resources methods has achieved great progress in recent years, it is not able to catch the pace of the development of ecology science. Based on the analysis of recent literatures about ecology development trend and Chinese traditional medicinal ecology methods, the progress of Chinese traditional medicinal ecology methods was reviewed, and future study trend was discussed.
Conservation of Natural Resources ; Ecology ; methods ; Medicine, Chinese Traditional ; Research ; trends ; Research Design

Objective:To construct and rescue enhanced green fluorescent protein(EGFP)-labeled EGFP-EV-A71 recombinant virus.Methods:pMD19T-SDLY107 full-length plasmid was used as a template, and 2A protease recognition sequence was added to the 5′end of the structural protein VP4. EGFP gene was amplified using the pEGFP-N1 plasmid as a template and inserted into the above-mentioned recombinant plasmid. RD cells were transfected to rescue the recombinant virus EGFP-EV-A71 after enzymatic digestion and in vitro transcription. The cell culture infectious dose 50% endpoint (CCID 50) was determined. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the virus replication level at different time points, and the virus replication curve was drawn to compare the replication ability of the recombinant virus and the parent virus. Lactate dehydrogenase (LDH) and cell proliferation (CCK-8) experiments were used to determine the cell injury rate and survival rate of infected cells, respectively. Results:Recombinant EV-A71 infectious cDNA clones containing specific restriction sites and EGFP were successfully constructed. Typical cytopathic effect and green fluorescence was observed. The qRT-PCR replication curve showed that the recombinant virus had similar replication kinetics to the parent virus.Conclusions:EGFP-labeled enterovirus type 71 recombinant virus EGFP-EV-A71 was successfully rescued.

Objective:To explore the effect of key amino acid mutations of 3D protein in enterovirus 71 on viral proliferation, and infer the mechanism of mutation affecting viral proliferation according to the tertiary structure model of 3D protein.Methods:The proliferation characteristics of the mutant strain that was constructed by site-directed mutagenesis and reverse genetics using the pMD19T-SDLY107-EGFP constructed from the fatal strain SDLY107 as a template were measured. Meanwhile the tertiary structure of the 3D protein was predicted, and then the possible mechanism of the mutation affecting the proliferation ability of the virus was speculated according to the functional characteristics of the domain of the 3D protein.Results:Two mutant viruses, eGFP-EV-A71 (S37N) and eGFP-EV-A71 (R142K), were successfully constructed by double enzyme digestion and viral gene sequencing. The proliferation rate of the two mutant strains in RD cells was significantly lower than that of the parent strains. The 3D protein tertiary structure prediction model showed that the 3D protein consisted of three domains: "Finger" , "thumb" and "Palm" , constituting a cupped right-handed structure. S37N and R142K are located in the "thumb" domain and " finger" domain, respectively. The "thumb" and "finger" domains have important effects on the activity of 3D polymerase and the stability of protein.Conclusions:Mutations at S37N and R142K sites of EV71 3D protein decrease the replication ability of EV71, and these two mutations may affect the proliferation of EV71 virus by changing the 3D protein polymerase activity and the interactions between multiple domains.