Effect Analysis of Artemisiae Annuae Herba and Chuanxiong Rhizoma in Treatment of Cerebral Malaria Based on Network Pharmacology / 中国实验方剂学杂志

Objective:To explore the reasonable combination of Artemisiae Annuae Herba and Chuanxiong Rhizoma in treatment of cerebral malaria and investigate its mechanism based on network pharmacology. Method:The traditional Chinese medicine systems pharmacology database and analysis platform （TCMSP） and SymMap were used to obtain all the chemical components of Artemisiae Annuae Herba and Chuanxiong Rhizoma and the action targets were screened to construct a component target protein-protein interaction （PPI） network. Target genes related to cerebral malaria were collected with use of GeneCards and DisGeNET databases. Common targets were screened by overlapping drug targets and disease targets， and protein-protein interaction network analysis was performed to get key targets. Gene ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were carried out to get main signaling pathways. Furthermore， the classical experimental cerebral malaria mouse model was used to detect survival curve， protozoanemia level， survival rate， experimental cerebral malaria （ECM） coma and behavior scores. RayBio^® cytokine antibody array was used to detect the expression level of cytokines in tissues and experiment was conducted for verification. Result:After combination of Artemisiae Annuae Herba and Chuanxiong Rhizoma， 23 active ingredients， 179 drug targets， and a total of 100 common targets of the drug and disease were obtained. GO functional analysis identified 59 items （<italic>P</italic><0.05）， involving cytokine activity， growth factor activity， immune response， etc. KEGG pathway analysis revealed 51 related signaling pathways. The experimental results showed that the combined use of Artemisiae Annuae Herba and Chuanxiong Rhizoma could significantly improve the clinical signs of ECM mice， such as survival state， coma and behavioral scores. In the detection of expression levels of related cytokines in mice， the expression levels of <italic>γ-</italic>interferon （IFN-<italic>γ）</italic>， interleukin-10 （IL-10）， IL-4， and IL-1<italic>β</italic> in the compatible drug combination drug were significantly higher than those in the model group （<italic>P</italic><0.05）， which was consistent with the overlapping core targets predicted by network pharmacology. Conclusion:Based on the network pharmacology analysis and<italic> in vivo</italic> experiment verification， this study confirmed the synergistic effect of the combination of Artemisiae Annuae Herba and Chuanxiong Rhizoma in the treatment of cerebral malaria， providing clear direction for further mechanism research， and a new possibility for the clinical intervention of cerebral malaria.

Effect and Mechanism of Shenlian Formula in Treatment of Atherosclerotic Cardiovascular Disease Based on Network Pharmacology / 中国实验方剂学杂志

Objective:To analyze active components， its targets and signaling pathways of Shenlian formula based on network pharmacology， and explore the molecular mechanism of Shenlian formula in the treatment of atherosclerotic cardiovascular disease （ASCVD）， in order to provide a basis for the rational interpretation of the prescription compatibility of Shenlian formula. Method:Major chemical compounds of the formula were obtained by SymMap and Systematic pharmacology database and analysis platform of Traditional Chinese Medicine （TCMSP）， its target proteins were obtained by SymMap and ETCM Databases， and the pathogenic genes responsible for of ASCVD were obtained by DisGeNET and GEO Datebases. Protein targets of drugs and pathogenic genes of diseases were overlapped to obtain predicted targets of Shenlian Formula for ASCVD. Proteins-proteins interactions （PPI） network was built through the String Datebase. The Cytoscape 3.6.0 was used to explore the key compounds and targets of Shenlian formula on ASCVD. Then gene ontology （GO） enrichment and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway were analyzed to screen out the key targets of Shenlian Formula. Rat I/R model was adopted as representative disease model of ASCVD for experimental verification. Result:There were 59 candidate compounds， 67 predicted targets and 29 key targets of Shenlian formula on ASCVD. Key targets mainly included cyclooxygenase 2 （PTGS2）， estrogen receptor 1 （ESR1） and TP53. GO analysis showed that the biological functions of potential genes of Shenlian formula in treatment of ASCVD were mainly related to apoptotic， nitric oxide biosynthetic process， response to estradiol， angiogenesis， inflammatory response and oxidative stress and acute-phase response. KEGG pathway enrichment results showed that the pathways of potential genes of Shenlian formula in treatment of ASCVD mainly involved TNF signaling pathway， phosphatidylinositol-3 kinase （PI3K）/ protein kinase B （Akt） signaling pathway， hypoxia induction factor-1 （HIF-1） signaling pathway and apoptosis. Among them， the regulatory effect of Shenlian formula on apoptosis may act on not only TP53， but also different signaling pathways of apoptosis respectively， thus playing a synergistic effect. <italic>In vivo</italic> experimentation confirmed that Shenlian formula could significantly reduce the myocardial infarction area， improve the myocardial histopathological changes， and especially reduce myocardial mitochondrial injury. Further analysis showed that Shenlian formula can significantly inhibit the expressions of activated proteins in mitochondrial apoptosis pathway. Conclusion:Anti-atherosclerosis traditional Chinese medicine Shenlian formula could effectively intervene ASCVD， and its effect on mitochondrial apoptosis of myocardial cells is one of its mechanisms in protecting myocardial ischemia-reperfusion injury.

Effect of Exogenous Nitric Oxide on Plant Hormones and Enzyme Content of Acanthopanax senticosus Seeds / 中国实验方剂学杂志

Objective: To investigate the effect of exogenous nitric oxide (NO) on breaking the dormancy of Acanthopanax senticosus seeds and the changes in endogenous hormones and enzymes,and provide a basis for breaking the dormancy as well as artificial cultivation of A. senticosus seeds.Method: Different concentrations (1,5,10,20 mmol·L-1) of sodium nitroprusside (NO donor) were used to treat the A. senticosus seeds, and then thermophilic stratification was conducted. The content changes of endogenous hormones such as gibberellin (GA3), abscisic acid (ABA),indolo acetic acid (IAA),indolo butyric acid (IBA) and salicylic acid (SA) at different stratification time (0, 30, 50, 80, 100,130 d) were tested by high performance liquid chromatography (HPLC). The activity change of its in vivo enzymes[catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), and malondialdehyde (MDA)] were tested by enzyme-labeled instrument.Result: In the seed germination process of A. senticosus,the contents of GA3,IAA,IBA,and SA were increased gradually,while the content of ABA was reduced gradually. The enzyme activities of POD and MDA were significantly reduced,and the enzyme activities of CAT and SOD were increased obviously. Exogenous NO could increase the seed germination rate and shorten the seed germination time. The effect of 20 mmol·L-1 sodium nitroprusside showed the most obvious effect and 10 mmol·L-1 SNP showed the weakest effect in promoting seed germination,showing an obvious "V" shape for changes.Conclusion: Sodium nitroprusside could promote the seed germination effect of A. senticosus, probably by increasing the content of hormones and enzyme in the stage of seed germination and improving the contents of endogenous NO during germination.

Analysis of miRNA differential expression in peripheral blood cells of primary immune thrombocytopenia patients / 中国实验血液学杂志

This study was purposed to clarify the difference of microRNA (miRNA) expression in the peripheral blood cells of patients with primary immune thrombocytopenia (ITP) and normal controls. Exqion miRCURY(TM) microarray was used to investigate differentially expressed miRNA of peripheral blood cells obtained from affected ITP patients and the healthy controls. Cluster analysis was used to identify miRNA expression profile between the ITP patients and the healthy controls. Real-time PCR was used for validation. The results showed that a total of 159 miRNA were found to be differentially expressed in ITP patients compared to the controls, with 79 up-regulated and 80 down-regulated. Based on these differentially expressed miRNA, a tree with clear distinction between the controls and ITP patients was generated by cluster analysis. Real-time PCR confirmed microarray analysis results. It is concluded that differentially expressed miRNA were found in the peripheral blood cells from ITP patients, which may be potential novel biomarkers for ITP as well as help to elucidate pathogenic mechanisms of ITP.

Cloning and expression of Hgp44 from Porphyromonas gingivalis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To clone and express the gene of Hgp44 in adhesin domains of gingipains from Porphyromonas gingivalis (Pg) and to purify the protein.</p><p><b>METHODS</b>The genomic DNA of Pg was isolated from PgATCC33277. The Hgp44 gene fragment was amplified by polymerase chain reaction (PCR) and then inserted into the cloning vector pMD18-T and sequenced. The correct fragment was linked with a prokaryotic expression vector pET-22b to construct the recombinant expression plasmid pET22b-Hgp44. The pET22b-Hgp44 confirmed by enzyme digestion was transformed into competent Escherchia coli (Ec) BL21 (DE3) cells. Expression of fusion protein was induced by isopropyl-β-D-thiogalactoside (IPTG), and purified by immobilized metal-chelating affinity chromatography (IMAC) using a Ni(2+) matrix column. SDS-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis were used to examine the fusion protein.</p><p><b>RESULTS</b>A 1 100 bp fragment was successfully amplified and verified by the agarose gel electrophoresis and sequencing. The generated recombinant expression vectors pET22b-Hgp44 were verified by enzyme digestion and agarose gel electrophoresis. The expression of fusion protein in Ec BL21 (DE3) cells was examined by SDS-PAGE and Western blotting analyses, and the data showed that the protein was 44 000 in size and expressed mostly in the form of inclusion body. The purification of fusion protein was achieved using Ni(2+) affinity chromatography. About 3.5 mg/L fusion protein was obtained.</p><p><b>CONCLUSIONS</b>Hgp44 was successfully expressed in the prokaryotic expression system and purified by IMAC using a Ni(2+) matrix column.</p>

Study on the optimal harvesting time of Rumex gmelini by analyzing the contents of principal components / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a method used for optimization of harvesting time and determine the best time for harvesting Rumex gmelini.</p><p><b>METHOD</b>An HPLC method was applied to determinate the contents of seven active constituents(resveratrol, polydatin, chrysophanol 1-glucoside, nepodin, emodin, chrysophanol and physcion)of R. gmelini at different development stage. The result was analyzed by principal component analysis.</p><p><b>RESULT</b>The accumulation of active constituents showed a regular pattern.</p><p><b>CONCLUSION</b>The best harvesting time of R. gmelini is early July.</p>