Potential application of Nardostachyos Radix et Rhizoma-Rhubarb for the treatment of diabetic kidney disease based on network pharmacology and cell culture experimental verification

Figure 1 Flow chart of network pharmacological prediction and experimental validation. GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; DKD: Diabetic kidney disease; PPI: Protein-protein interaction; BP: Biological process; CC: Cell composition; MF: Molecular function; IC₅₀: Half maximal inhibitory concentration.

Figure 2 Identification of drug and disease targets and protein-protein interaction enrichment analysis. A: Drug and disease targets; B: Intersection of drug and disease targets; C: Drug-disease-target network diagram; D: Protein-protein interaction (PPI) network diagram of intersection genes of Nardostachyos Radix et Rhizoma-rhubarb drug pair (NRDP) and disease; E: PPI network containing 73 intersection targets and 10 core targets constructed using Cytoscape; F: Selected core targets and active components of NRDP were analyzed. Each node’s color denotes the degree.

Figure 3 Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. A: Gene Ontology enrichment analysis; B: Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis; C: Enriched core genes in the AGE-RAGE signaling pathway; D: Correlation between AGE-RAGE signaling pathway and apoptosis.

Figure 4 Molecules docking. A: Docking results between Nardostachyos Radix et Rhizoma-rhubarb drug pair components and core target molecules; B: Heat map of molecular docking showing the binding energy of core target molecules.

Figure 5 Effect of NardostachyosRadix etRhizoma-rhubarb drug pair on cellular phenotype of TCMK-1 cells. A and B: Nardostachyos Radix et Rhizoma-rhubarb drug pair (NRDP) reduces the activity of TCMK-1 cells; C: Effect of NRDP on the cell cycle of TCMK-1 cells; D: Effect of NRDP on apoptosis of TCMK-1 cells. CON: Control group; MOD: Model group; NRDP: Nardostachyos Radix et Rhizoma-rhubarb drug pair; IC₅₀: Half maximal inhibitory concentration. ^aP < 0.001, ^bP < 0.001, ^cP <0.01.

Figure 6 Effect of NardostachyosRadix etRhizoma-rhubarb drug pair on AGE-RAGE signaling pathway in TCMK-1 cells. A, B, C, and E: Protein expression of BAX, BCL-2, Caspase9, and Caspase3; D and F: Expression of STAT3 and p-STAT3 protein. CON: Control group; MOD: Model group; NRDP: Nardostachyos Radix et Rhizoma-rhubarb drug pair. ^aP <0.001, ^bP < 0.001, ^cP <0.01, ^dP < 0.05, ^eP <0.05.

Figure 7 Mechanism of NardostachyosRadix etRhizoma-rhubarb drug pair in treatment of diabetic kidney disease. NAGE: Receptor for advanced glycation end products; ROS: Reactive oxygen species.