Molecular Mechanism of Resveratrol Against NAFLD Based on Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo predict the molecular mechanism of resveratrol against non-alcoholic fatty liver disease （NAFLD） based on network pharmacology and molecular docking and verify the results on the liver cell model induced by PM2.5 exposure. MethodThe targets of resveratrol were screened out from Traditional Chinese Medicine System Pharmacology Database and Analysis Platform （TCMSP）， PubChem， DrugBank， and SwissTargetPrediction， and the potential targets of NAFLD were retrieved from Comparative Toxicogenomics Database （CTD）， DisGeNET， GeneCards， and Online Mendelian Inheritance in Man （OMIM）. Then the common targets were obtained. STRING 11.5 was used to construct the protein-protein interaction （PPI） network of the common targets. Cytoscape 3.8.2 was used to plot the “target-pathway” network， and the core modules and key targets were selected. Metascape was adopted for Gene Ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） enrichment analyses of common targets. SYBYL-X 2.0 was used for molecular docking of resveratrol to key targets. Finally，cell apoptosis and the expression of apoptosis-related proteins were detected by flow cytometry and Western blot in the PM2.5-exposed human liver cell line （HepG2）. ResultA total of 115 common targets of resveratrol and NAFLD were obtained. The key targets included tumor necrosis factor （TNF）， B-cell lymphoma-2 （Bcl-2）， and cysteinyl aspartate-specific protease-3（Caspase-3）. As revealed by KEGG enrichment analysis， 174 signaling pathways， represented by the apoptosis and TNF signaling pathways， were obtained. Molecular docking results showed that resveratrol had strong binding activities to Bcl-2 and Caspase-3. Furthermore，the results of flow cytometry and Western blot demonstrated that resveratrol inhibited cell apoptosis of PM2.5-exposed HepG2 cells by regulating the protein expression of Bcl-2 and Caspase-3. ConclusionResveratrol can treat NAFLD in a multi-pathway and multi-target way. It mainly inhibits liver cell apoptosis by affecting the expression of Bcl-2 and Caspase-3， which provides a theoretical basis for the follow-up research on the anti-NAFLD mechanism of resveratrol.

Effects of α-pinene extracted from pine needle on expression of miR-221 and its potential target genes in human hepatocellular carcinoma cells / 中国中药杂志

To investigate the anti-hepatoma mechanism of α-pinene, HepG2 cell was treated with α-pinene and the change of cell cycle was examined by flow cytometry. The expression of miR-221, which was related the regulation of G₂/M phase, was detected by quantitative Real-time PCR. Meanwhile, TargetScan and other online bioinformatics methods were used to analyze and predict the target genes of miR-221, then the expression level of related target genes were detected by quantitative Real-time PCR. The results showed that α-pinene inhibited the proliferation of HepG2 cells in dose-dependent manner. It was also proved that HepG2 cells were arrested at G₂/M phase by α-pinene (P<0.05). The expression of miR-221 was down-regulated in α-pinene treated HepG2 cell. The bioinformatics analysis showed that CDKN1B/P27 and CDKN1C/P57 may be the protential targets of miR-221 and both of them were significantly up-regulated（P<0.001,P<0.05）by α-pinene treatment. According to these results, it was believed that α-pinene may inhibit the proliferation of hepatocellular carcinoma cells through arrest the cell at G₂/M phase, which may be associated with the down-regulate of the miR-221 expression and up-regulate of the CDKN1B/P27 and CDKN1C/P57 expression.

To investigate pathogen of hand, foot and mouth disease in Shenzhen in 2008 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate EV71 and CA16 pathogen of HFMD in Shenzhen in 2008, and to provide the evidence for the prevention and treatment HFMD.</p><p><b>METHOD</b>Using RT-PCR technology to detect the EV71 and CoxA16 genes of 307 samples HFMD; sequencing the purified PCR products from 14 samples. Using ClustalW2 online analysis software for sequence and phylogenetic analysis of enterovirus 71.</p><p><b>RESULT</b>Percentage of positive EV71 from different samples is shown as follows respectively: positive EV71 from stool samples is 24.4% (75/307), from throat swab--7.8% (24/307), from peripheral blood--12.5% (1/8). Percentage of positive CoxA16 is shown as follows respectively: positive EV71 from stool samples is 13.8% (28/203), from throat swab-11.0% (20/181). Among all the 307 samples, three are positive for both EV71 and CoxA16. EV71 and CoxA16 are not detected in the samples of cerebrospinal fluid.Comparative analysis of nucleotide sequences of EV71 with those of strains BrCr and 11 deposited in GenBank demonstrated numerous disparities from 8 samples, but residue 595 from 2 samples and residue 658 from 1 sample are variable. The phylogenetic analysis based on VP1 region demonstrates that strains from 2 samples has the nearest genetic relationship with anhui strains, the farthest with BrCr and SHH02-6, SHZH02-40, SHZH03-58 strains, also strains from other 12 samples have the farthest genetic relationship with them. The genotypes A, B and C were classified as proposed by Brown et al. (1999). The EV71 from 14 samples were the member of genotype C.</p><p><b>CONCLUSION</b>EV71 among the pathogen of HFMD in Shenzhen in 2008 was majority. These EV71 may belong to the same genegroup with Anhui predominant strains.</p>

Forecast on shelf life of Chinese Angelica based on its chemical components variation / 中国中药杂志

<p><b>OBJECTIVE</b>To predict the shelf life of Chinese Angelica by investigating the stability of its active components.</p><p><b>METHOD</b>The classic homeothermia method was used for forecast the shelf life, and HPLC was applied for quantitative determination of chemical compounds in Chinese Angelica.</p><p><b>RESULT</b>The correlation equations, thermal constant at ambient temperature (k(25 degrees C)) and shelf life (t(0.9)) for coniferyl ferulate and Z-Ligustilide were lgk = -5152. 1/T + 13.8 (r = 0.9985), 3. 33 x 10(-4), 13 d and lgk = -4064.6/T + 10.4 (r = 0.9997), 5.91 x 10(-4), 7 d, respectively.</p><p><b>CONCLUSION</b>The shelf life of Chinese Angelica has been established, which suggests that it is very important for ensuring the safety and efficacy of Chinese crude drug.</p>

Effect of hepatitis B virus C protein on function of natural killer cell in NK-92 cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the influence of hepatitis B virus C protein on the function of natural killer cell.</p><p><b>METHODS</b>Recombinant eukaryotic expression plasmid pHBI-CMV-HBC was constructed and confirmed by double restrictive enzyme digestion and DNA sequencing analysis. Then the recombinant plasmid was transfected into NK-92 cells with lipofectamine encapsuled. The transfected NK-92 cells containing expressive HBV C protein was confirmed by Western Blot analysis. ELISA was employed to determine the IFN-gamma level secreted by NK-92 cells. And finally the cytotoxicities of NK cells were analysed by MTT colorimetry, with the hepatoblastoma cell line (HepG2) as target cell.</p><p><b>RESULTS</b>Western blotting confirmed the expression of HBV C protein in the NK-92 cells transfected with pHBI-CMV-HBC. NK cytotoxicities and IFN-gamma secretion level of NK-92 cells transfected with recombinant plasmid significantly increased compared to control NK-92 cells transfected with blank plasmid (P < 0.01) and untransfected NK-92 cells(P < 0.01).</p><p><b>CONCLUSION</b>Transient expression of HBC can increase IFN-gamma secretion and cytotoxicities of NK-92 cells.</p>

Study of the clinical and laboratory features of hand-foot-mouth disease / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the clinical and laboratory features of the mild and severe hand-foot-mouth diseases (HFMD) in Shenzhen in 2008.</p><p><b>METHODS</b>145 cases were observed in East-Lake Hospital and Shenzhen Children's Hospital. Of the 145 cases, 124 mild cases and 21 severe cases were involved.All the clinical data and laboratory findings were collected and summarized. After collection of the acute and convalescent consecutive stools and peripheral bloods from the patients with HFMDI, EV71 genes were amplified from these samples by RT-PCR. Enterovirus 71 were cultured and isolated using Vero cell line and R&D cell line.</p><p><b>RESULTS</b>The WBC counts and blood glucose levels of the severe cases were significantly elevated, but the ages of the severe ones significantly decreased compared with those of the mild cases (P < 0.05). EV71 genes could be detected by RT-PCR with 35% positive rate in mild cases and 67% in severe cases. The EV71 gene detection rate of the severe cases was significantly increased in contrast to that of the mild ones. The EV71 were isolated and cultured from the stools of 9 patients, one specimens from the dead's stool. Two severe cases died of neurogenic pulmonary edema and brain-stem encephalitis.</p><p><b>CONCLUSIONS</b>EV71 mainly contributes to HFMD and is responsible for death of some severe cases. High fever, less rash, elevated white blood cell counts and blood glucose concentrations as well as age less than 4 years old should be used for prediction of severe cases.</p>

Expression of recombinant VP1 protein of enterovirus 71 and development of serological assay for detection of EV71 infection / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To obtain a recombinant purified Enterovirus 71 VPI protein and establishment of an early, rapid and accurate serological ELISA (enzyme-linked immunosorbent assay) for detection of EV71 infection.</p><p><b>METHODS</b>VP1 gene was amplified by PCR and clonel into pET-21b (+) vector, the positive recombinant plasmid were transformed into E. coli BI21(DE3), and was induced with IPTG, the recombinant protein by SDS-PAGE and Western Blot assays. Finally, the recombinant purified VP1 protein was used as a coated antigen for detection of serum anti-IgM and IgG against EV71 by ELISA.</p><p><b>RESULTS</b>The purified VP1 was obtained, and it can be recognized by sera of patients with EV71 infection associated with hand-foot-mouth disease. The A values of anti-EV71 IgM and IgG were significantly elevated as compared to healthy objects and HFMD patients without EV71 infection (P < 0.05). The sensitivity and specificity of IgM to EV71 were 73% and 77% compared with the RT-PCR results, respectively;and those of IgG being 82% and 83%, respectively.</p><p><b>CONCLUSIONS</b>The recombinant protein VP1 was produced and purified, and it was proved to have a good antigenicity and could be used to develop a serological diagnosis kit for EV71 infection in the future.</p>