Network Pharmacology Prediction and Experimental Validation of Trichosanthes-Fritillaria thunbergii Action Mechanism Against Lung Adenocarcinoma

Traditional Chinese Medicine, We'll speak of the characteristics of multicomponent of the target. Which is the very basic box for Network and Biological research. We did a demonstration of the technology that will reduce the threshold.

Network Pharmacological is systematic analytical technology, builds the tools of interaction network work of multifactors. It can effectively predicts the single pathway of Traditional Chinese Medicine in The data of Network Pharmacological comes from the database, although it can't predict the relationship to drugs, proteins, and this it's still need to be verified by the by subsequent experiments. To begin open the HERB Database and use Gua Liu and Zhebimu as keywords to obtain the components of the two drugs.

Download the list and the canonical SMILES structure of the related components of the two drugs. Now determine whether the obtained component is active by including components with oral bioavailability and drug-like values in the HERB Group Database. If the component has no oral bioavailability and drug-like values, input the component into the Swiss ADME Database to obtain the information on each component.

Include components with high GI absorption and at least two yes drug-like values as active components. To predict the target of the active components open the HERB Database. Search and copy the canonical SMILES structures of the active components.

Then open the similarity ensemble approach and paste the canonical SMILES structures of the active components into the search box. Click on try SEA'to obtain the target key target name P-value, and maximum TC of each active component. Copy the data into a spreadsheet and use the spreadsheet files filtering function to filter the active components targets by target key.

Copy all the targets to a spreadsheet and remove the duplicates to obtain the drug targets. Now open the gene cards database and online Mendelian inheritance in man to predict disease targets. Use Lung Adenocarcinoma'as the keyword to obtain the disease targets of the Lung Adenocarcinoma.

Download the spreadsheets of the disease targets and delete the repeated targets, to obtain the Lung Adenocarcinoma targets. Construct a drug component disease target network by copying the lung adenocarcinoma related targets and drug targets in into the same column, in a new spreadsheet. Use the data identify duplicates function, in the toolbar to obtain intersection targets of the Lung Adenocarcinoma related targets and the Trichosanthes Fritillaria Thunbergia active component related targets.

Open Cytoscape 3.8.0. Click on file in the menu bar and select import. Followed by network from file.

To import the spreadsheet file, optimize the size and color of the network nodes through the style bar in the left control panel. Use the analyze network function for the network topology analysis. Click on tools in the menu bar and select analyze network.

On the table panel, click on the degree in the title bar to arrange the components by degree in descending order. Take the top 10 components and targets as the main active components and core targets. To construct the PPI network and screen the core proteins.

Open the string database and paste the text format list of the potential targets of Trichosanthes Fritillaria Thunbergia, against Lung Adenocarcinomas into the list of names'dialogue box. Then select homosapiens in organisms. Click search and select continue buttons, when the results are available, click settings And tick high confidence 0.700 In basic settings, under minimum required interaction score tick high disconnected nodes in the network, in advanced settings.

Then click the update button. Next, click on exports in the title bar and download the short tabular text of the PPI relationship in TSV format. Open Cytoscape 3.8.0.

Click on file, select, import, followed by network from file to import the TSV format file for visual analysis. Use the network analyzer function to conduct the topological analysis. Optimize the size and color of the network nodes through the style bar in the left control panel.

Perform KEGG enrichment analysis by opening the Medscape platform, pasting the text format list of the potential therapeutic targets into the dialogue box, and then clicking on the submit'button. Tick Homo sapiens in both input as species'and analysis as species'and then click on the custom analysis button. Select enrichment.

Tick only the KEGG pathway and then click on enrichment analysis. Once the progress bar reaches 100%click the analysis report page orange button for the enrichment results. Click on all in one zip file'to download the enrichment result and then open the _FINAL_GO'csv file in the Enrichment_GO'folder to obtain the result.

Open R-Software and type install package GG plot two'and library GG Plot two'in R For the installation of the GG plot two R package. Press enter to run the KEGG Visualization program. To detect cell viability, digest the logarithmic grow phase A 549 cells with one milliliter of 0.25%Trypsin for one minute at 37 degrees Celsius.

Add one milliliter of DMEM Complete Medium to neutralize the Trypsin and gently blow it to promote cell shedding. Then centrifuge the mixture to obtain the cell pellet and resuspend the obtained cells, using DMEM Complete Medium. Add the cell suspension to a Hemocytometer and count using an automated cell counter, dilute it to five times 10 to the fourth cells per milliliter, using DMEM complete medium Now dissolve one gram of Trichosanthes Fritillaria Thunbergia water extract in 10 milliliters of PBS and filter sterilize it through a 0.22 micrometer filter.

Dilute the mixture to different concentrations using PBS. Plate 100 microliters of the diluted cells into each well of a 96 well plate. After cell adherence, add one microliter of Trichosanthes Fritillaria Thunbergia water extracts of different concentrations to adjust the concentration of each well.

Discard the original medium after 24 hours of culture and add 100 microliters of DMEM. Basic medium for further incubation for two hours at 37 degrees Celsius and 5%carbon dioxide. At the end of the incubation, add 20 microliters of MTS solution and incubate the cells for another one hour.

Transfer the incubated mixture to a different plate. Using a microplate reader, measure the absorbence at a 490 nanometers wavelength and calculate the cell viability. Dilute the logarithmic growth phase A 549 cells to five times 10 to the fifth cells per milliliter.

Add two milliliters of the cell suspension to a six well plate and grow for 12 hours. After obtaining different PBS dilutions of Trichosanthes Fritillaria Thunbergia water extract as demonstrated earlier, add 20 microliters of the PBS solution to the blank control group and 20 microliters of the various dilutions of Trichosanthes Fritillaria Thunbergia water extract to different concentration group. After 24 hours of intervention, discard the supernatant and clean the cells with PBS three times.

Add 250 microliters of RIPA buffer to each well and lyse the cells for 30 minutes. Collect the lysate for centrifugalization and obtain the supernatant. The drug component disease target interaction network of Trichosanthes Fritillaria Thunbergia against Lung Adenocarcinoma is shown.

In the interaction network, The top 10 active components obtained are the key active components of the action of Trichosanthes Fritillaria Thunbergia in treating Lung Adenocarcinoma. The PPI network in included 122 functional proteins and 210 interaction relationships. The top 10 core proteins are mainly involved in neovascularization, cell proliferation, apoptosis and cell membrane transport.

Of the top 20 pathways ranked by KEGG, the PI3K-AKT signaling pathway, Rap1 signaling pathway, Phospholipase-D signaling pathway, and MAPK1 signaling pathway are closely associated with lung cancer, among which the PI3K-AKT pathway ranked the first. Trichosanthes Fritillaria Thunbergia extracts at concentrations over 400 micrograms per milliliter could inhibit cell proliferation and the inhibition effect on A-549 cells at concentrations up to 800 micrograms per milliliter, was close to half inhibitory concentration. The intervention of Trichosanthes Fritillaria Thunbergia extracts caused no significant change in AKT protein expression in each group.

However, p-AKT Serine-473 expression was inhibited, and showed a dose dependent effect. The critical components of Trichosanthes Fritillaria Thunbergia were molecularly docked with the key proteins of the PI3K AKT pathway, and the results suggested the binding energies of Diosmetin and Kaempferol with AKT1 were less than minus seven, indicating strong binding activity. The most important thing is to have achieved active ingredients on the targets of Traditional Chinese Medicine.

As well as the targets of which is the core of Network Pharmacological.