[Study on mechanisms of "Huangqi-Shengdihuang-Shanyao" in treating type 2 diabetes based on network pharmacology and experimental verification].

This study aims to explore the mechanisms of "Huangqi(Astragali Radix)-Shengdihuang(Rehmanniae Radix)-Shanyao(Dioscoreae Rhizoma)"(HSS) in the intervention of type 2 diabetes mellitus(T2DM). The active ingredients and targets of the triangular herb HSS were searched from the TCMSP, BATMAN-TCM, and DrugBank databases. The disease-related targets of T2DM were searched from the PharmGkb, GeneCards, OMIM, and TTD. The intersection targets of HSS and T2DM were obtained, and the protein-protein interaction(PPI) analyses of the intersection targets were performed by using the STRING and Cytoscape software. The Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) signaling pathway enrichment analyses for the intersection targets were performed through R software. After continuous intragastric administration of HSS formula granules for one week, blood was collected from the abdominal aorta of SD rats to obtain herb-containing serum. An insulin resistance-HepG2(IR-HepG2) cell model was induced by insulin solution combined with high glucose medium. The groups were divided into a blank group, a control group, a model group, a low-dose HSS group, a medium-dose HSS group, and a high-dose HSS group. The cellular viability, glucose consumption, glycogen content, malondialdehyde(MDA), superoxide dismutase(SOD), and reactive oxygen species(ROS) were detected using the corresponding kits. The mRNA expression of phosphoinositide 3-kinase(PI3K), protein kinase B(Akt), and glycogen synthase kinase-3β(GSK-3β) was assayed through real-time PCR, and the protein expression of PI3K, Akt, phosphorylated(p)-Akt, GSK-3β, and p-GSK-3β was assayed through Western blot. A total of 10 903 disease-related targets of T2DM were obtained, and 29 active ingredients of HSS that may act on T2DM were screened out. These active ingredients were associated with 196 T2DM-related targets. Quercetin, kaempferol, and formononetin were the active ingredients that act on the largest number of T2DM targets. GO analysis of the intersection targets enriched 2 396 biological processes, 95 cellular components, and 198 molecular functions. KEGG analysis of the intersection targets enriched 175 pathways including PI3K/Akt signaling pathway, mitogen-activated protein kinase(MAPK) signaling pathway, and advanced glycation end-products/receptor for advanced glycation end-products(AGE/RAGE) signaling pathway. A total of 17 core proteins were obtained through the topology analyses of the PPI network. Low-, medium-, and high-dose HSS-containing serum had no significant effect on the proliferation of IR-HepG2 cells. HSS-containing serum could increase glucose consumption and glycogen content, reduce the MDA and ROS levels, and increase the SOD activity in IR-HepG2 cells. Additionally, the HSS-containing serum significantly up-regulated the PI3K mRNA expression, down-regulated the GSK-3β mRNA expression, up-regulated the PI3K, p-Akt, and p-GSK-3β protein expression, and down-regulated the GSK-3β protein expression, all in a dose-dependent manner. The mechanism of HSS acting on T2DM is a complex network with multiple targets and multiple pathways. HSS-containing serum could effectively ameliorate the insulin resistance of IR-HepG2 cells, and its therapeutic mechanism may be related to the activation of the PI3K/Akt/GSK-3β signaling pathway and the improvement of antioxidant stress resistance of model cells.