To begin, open the traditional Chinese medicine systems pharmacology database and analysis platform. Enter the medicinal composition for Jiawei Shengjiang San, or JWSJS, and apply the screening criteria. Then using the database, retrieve the targets corresponding to the ingredients.
Obtain the active ingredients of Bombyx batryticatus and Cicadidae Periostracum in the traditional Chinese medicine and chemical composition databases. After selecting compounds with chemical abstract service numbers, download 2D structure diagrams of active ingredients from PubChem. With SwissADME, screen the components with high gastrointestinal absorption as drug similarity whose two items or more were yes.
Import these into the database for target protein prediction. In the UniProt, set the status as reviewed and species as human and standardize the targets. Search diabetic nephropathy, or DN, through various databases and obtain the targets.
After combining and de-duplicating. Using our 4.2.0 software, screen the common targets of JWSJS and DN to draw Venn diagrams. Import the active ingredients and potential targets of JWSJS into Cytoscape 3.8.0 software to build a drug ingredient target network diagram that visualizes the connection between drugs, ingredients, targets, and diseases.
To analyze the intersecting genes in STRING platform, set the species to Homo sapiens and the minimum required interaction score to more than 0.9 for constructing the network using a multiple protein analysis mode. Using Cytoscape 3.8.0, analyze the network topologically and calculate the betweenness centrality, closeness centrality, degree centrality, eigenvector centrality, local average connectivity-based method, and network centrality values for each node. Then obtain the IDs of the intersection targets with orghs.eg.
db package. Using the cluster profiler org.hs.eg. db, enrichplot and ggplot2 packages, perform enrichment analyses.
Screen for functional gene ontology enrichment analysis on the top 10 biological hits with a corrected p-value less than 0.05, and select top 30 pathways with the highest enrichment for Kyoto Encyclopedia of Genes and Genomes Analysis. Search in the PubChem database to obtain the SDF file of the 2D structure of JWSJS core components. Using ChemBio3D Ultra 14.0 software, generate and optimize its 3D structure.
Save it in MOL2 format to use it as a ligand file. Find and download the PDB format of the 3D structure of the core targets from the research collaboratory for structural bioinformatics protein data bank database. Using PyMOL 2.4.0 software, remove water molecules and ligands from the protein structure and save it as PDB receptor file.
Import the receptor protein file into AutoDock Tools 1.5.7 software for hydrogenation and convert both receptor protein and small molecule ligand into PDBQT format. Set active pocket for receptor protein with spacing coefficient set to one. Using AutoDock Vina 1.2.0 for molecular docking, calculate the binding energy.
Visualize the docking results using PyMOL 2.3.0 and LigPlot 2.2.5 software. Conduct molecular dynamic or MD investigations on a trio of the most promising derived ligand complexes, and use SBC aqueous environment with 0.15 molar sodium chloride. Initiate an energy minimization phase for 100 picoseconds using Desmond default parameters.
Ensure stable temperature and pressure of 26.85 degrees Celsius and 1.01325 bar across all production systems through the Nose-Hoover chain and Martyna-Tobias-Kline methodologies. Execute the MD simulation for 100 nanoseconds with a time step of two femtoseconds, recording the atomic coordinates at every 100 picoseconds. Open the simulation interactions diagram or SID module and load the simulation result data files into it.
Once the analysis is complete, view and interpret the results within the SID module interface. Inject the model group animal with an intraperitoneal injection of streptozotocin. After 72 hours, test the blood sugar levels through tail vein blood sampling.
Observe the histopathological changes in the rat kidney to confirm the successful DN model. Then administer appropriate drugs to the rat via oral gavage once daily. Collect blood samples from the anesthetized rat's abdominal aorta.
Finally, after euthanizing the rat, collect the kidneys for biochemical and microscopic analyses. The periodic acid-Schiff staining showed that the model group had increased glomerular volumes, thickened basement membrane, and increased mesangial matrix compared to the normal group. These pathological manifestations were significantly alleviated in each drug administered group.
Transmission electron microscopy indicated that the glomerular basement membrane in the model group was thickened versus the normal group. Microscopic manifestations of rats in each administration group were relieved to varying extents versus the model group. Compared to the model group, there was a significant reduction in the expression of p-EGFR, p-MAPK3/1, and BAX, and upregulation of BCL-2 expression in rat kidney tissues of each dosing group to varying degrees.
