Mechanism of Indole-3-Methanol in Treatment of Obesity Based on Network Pharmacology and in Vitro Experiment / 中国实验方剂学杂志

ObjectiveTo preliminarily predict the targets and signaling pathways of indole-3-methanol in the treatment of obesity based on molecular docking technology and network pharmacology， and then verify the prediction results by the experiment in vitro. MethodThe pharmacological targets of indole-3-methanol were obtained from SwissTargetPrediction and literature review. Obesity-related targets were obtained from Online Mendelian Inheritance in Man （OMIM）， GeneCards， and Comparative Toxicogenomics Database （CTD）. The protein-protein interaction network of the targets of indole-3-methanol and obesity was built by STRING. Cytoscape 3.8.2 was used for target screening. Gene ontology （GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment were performed for the common targets shared by obesity and indole-3-methanol in DAVID 6.8. AutoDock Vina 1.1.2 was employed to perform the molecular docking between indole-3-methanol and disease targets. Finally， the in vitro experiment was carried out to verify the anti-obesity effect of indole-3-methanol. ResultIndole-3-methanol and obesity shared 80 common targets， which included matrix metalloproteinase （MMP）-9， Janus kinase （JAK） 2， etc. KEGG enrichment predicted that indole-3-methanol mainly acted on tumor necrosis factor （TNF）， vascular endothelial growth factor （VEGF）， tyrosine kinase receptor 2 （ErbB2）， and epidermal growth factor receptor （EGFR） signaling pathways in the treatment of obesity. Molecular docking showed that indole-3-methanol had good binding activity with fat mass and obesity-associated protein （FTO）. The results of Western blot， MTT assay， and oil-red O staining showed that indole-3-methanol down-regulated the expression of FTO in 3T3-L1 cells （P<0.05）. ConclusionIndole-3-methanol may treat obesity by down-regulating the expression of FTO protein and further inhibiting adipocyte proliferation. This study provides an experimental basis for deciphering the anti-obesity mechanism of indole-3-methanol.

Mechanism of Orcinol Glucoside in Treatment of Osteoporosis： An Exploration Based on Network Pharmacology and in Vitro Validation / 中国实验方剂学杂志

ObjectiveTo predict the therapeutic targets and related signaling pathways of orcinol glucoside （OG） in the treatment of osteoporosis by network pharmacology， and further clarify its mechanisms based on molecular docking and in vitro cell model. MethodThe pharmacological targets of OG were obtained from Similarity ensemble approach （SEA） and SwissTargetPrediction， and the targets related to osteoporosis from DisGeNET and GeneCards. The cross-analysis was conducted to screen the common targets between OG and osteoporosis. STRING was used to construct the protein-protein interaction （PPI） network， followed by topology analysis using CytoNCA plug-in of Cytoscape 3.7.2 to screen out the core targets. The obtained common targets were subjected to gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） analysis by g：Profiler. AutoDock Vina was utilized for molecular docking， and the in vitro cell experiments were then carried out for verifying the mechanism of OG in treating osteoporosis. ResultA total of 73 targets related to OG and osteoporosis were harvested，among which 14 were proved to be key targets by topological analysis. GO and KEGG functional enrichment analysis yielded 259 cell biological processes， mainly involving organonitrogen compound metabolic process， cell population proliferation， protein metabolic process， regulation of response to stress， and response to chemicals. Its mechanism of action might be related to advanced glycation end-product （AGE）-AGE receptor （RAGE） signaling pathway， interleukin-17 （IL-17） signaling pathway， and phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. Molecular docking indicated that the binding energies of OG to Cyclin D1 （CCND1） and cyclin-dependent kinase 4 （CDK4） were the lowest and similar. The results of flow cytometry showed that compared with the normal group， OG group exhibited decreased proportion of cells in G0/G1 phase （P<0.01） and decreased proportion of cells in S phase （P<0.01）. As demonstrated by Western blot， compared with the normal group， OG up-regulated the protein expression levels of Cyclin D1 and CDK4 （P<0.05， P<0.01）. ConclusionOG alleviates osteoporosis via multiple targets and multiple pathways. It may exert the therapeutic effects by increasing Cyclin D1 and CDK4 protein expression to change cell cycle and promote cell proliferation.

Mechanism of Arnebia euchroma Against Melanoma： An Exploration Based on Network Pharmacology and Experimental Verification / 中国实验方剂学杂志

ObjectiveTo preliminarily predict the active components， action targets， and signaling pathways of Arnebia euchroma in the treatment of melanoma based on network pharmacology and molecular docking， and to verify its possible mechanism of action in in vitro experiments. MethodThe active components and related targets of A. euchroma were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）SwissTargetPrediction and literature， and the targets related to melanoma from the GeneCards， Online Mendelian Inheritance in Man （OMIM）， and Comparative Toxicogenomics Database （CTD）. Following the construction of the protein-protein interaction （PPI） network of active components and related targets of A. euchroma and melanoma-related targets using STRING， Cytoscape 3.8.2 was used for screening and analyzing the nodes in the network of A. euchroma against melanoma. The intersections were subjected to gene ontology （GO） and Kyoto encyclopedia of genes and genomes （KEGG） enrichment analysis using DAVID 6.8. Acetyl alkannin， the active component in A. euchroma， was docked to the target by AutoDock Vina 1.1.2. The in vitro experiments were then carried out to verify the anti-melanoma effect of A. euchroma. ResultA total of 271 common targets of A. euchroma and melanoma were harvested， among which 23 were key targets， including matrix metalloproteinase-9 （MMP-9） and Janus kinase 2 （JAK2）. As revealed by KEGG enrichment analysis， A. euchroma mainly acted on Janus kinase/signal transduction and activator of transcription （JAK/STAT）， tyrosine kinase receptor （ErbB）， and vascular endothelial growth factor （VEGF） signaling pathways to resist melanoma. According to molecular docking， acetyl alkannin exhibited a good docking activity with JAK2， STAT3， VEGF， MMP-9， and E-cadherin receptors. The results of Western blot and Real-time quantitative polymerase chain reaction （Real-time PCR） showed that acetyl alkannin at different doses inhibited the protein and gene expression of JAK2， STAT3， VEGF， MMP-9， and E-cadherin in A375 cells （P<0.05）. ConclusionA. euchroma alleviates melanoma via multiple targets and multiple pathways， and it may exert the therapeutic effects by affecting the expression of such key target proteins as JAK2， STAT3， VEGF， MMP-9， and E-cadherin and inhibiting the invasion and metastasis of melanoma cells. This study has provided an experimental basis for the treatment of tumor with A. euchroma.

In silico designing of siRNA targeting PB 1 gene of Influenza A virus and in vitro validation.

Genomic variability makes Influenza A virus (IAV) ‘the least susceptible’ to existing vaccines or anti-influenza drugs. siRNA targeting viral gene silents the gene by cleaving mRNA. Present study aimed to develop siRNA targeting polymerase basic 1 (PB1) gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region of PB1 gene of IAV strains. Total 147 strains worldwide and 42 Indian strains, when aligned, showed seven sets of conserved regions (> 30 bp stretch and < 5% mismatches). To choose the most efficient siRNA, three levels screening method was developed. Finally one pair of siRNA was chosen due to its unique position in conserved region. siRNA efficacy was confirmed in vitro on Madin Darby Canine Kidney (MDCK) cell line propagating two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/ 2011(H3N2)] and Influenza A/pandemicH1N1 [A/India/LKO2151/2012(H1N1)]. The longest ORF was targeted by the selected siRNA, which showed 57 % inhibition in replication of Influenza A/pdmH1N1 and 60.6 % inhibition in replication of Influenza A/H3N2 at 72 hpi and 48 hpi respectively on MDCK cell line. This study shows that siRNA targeting PB1 may be moderately effective in controlling IAV replication so can be used as anti-IAV therapeutic agent.