Integrating serum pharmacochemistry and network pharmacology to elucidate the bioactive constituents and pharmacological mechanisms of Simiao Yong'an decoction in amelioration of insulin resistance in type 2 diabetes mellitus via PI3K/AKT pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Simiao Yong'an Decoction (SMYAD) is a classic heat-clearing and detoxifying formula that has been applied for centuries in Traditional Chinese Medicine (TCM). It is clinically employed to treat type 2 diabetes mellitus (T2DM) and associated complications, yet its potential active components and pharmacological mechanism of action remain incompletely defined.

OBJECTIVE: This study employed an integrated strategy combining serum pharmacochemistry and network pharmacology, with experimental validation in vivo, to systematically elucidate the potential active components of SMYAD and their mechanisms in ameliorating insulin resistance in T2DM.

MATERIALS AND METHODS: The chemical constituents of SMYAD in vitro and its serum migrant components in both normal and T2DM model rats were identified by UPLC-Q-Exactive Orbitrap-MS/MS, with differences between the two groups compared. By focusing on the differential components, network pharmacology was employed to screen for core targets and key pathways. The pharmacodynamic effect of SMYAD alleviating T2DM was demonstrated by in vivo experiments. Furthermore, molecular docking and molecular dynamics (MD) simulations were employed to predict the interaction patterns between the pathologically specific serum migrant components of SMYAD and the key targets, with surface plasmon resonance (SPR) experiments confirming their binding affinities, thereby elucidating the pharmacodynamic basis at the molecular level.

RESULTS: In the analysis of SMYAD's in vitro components, a total of 121 constituents were identified. In the serum migration study, 16 prototype components were detected in normal rats and 21 in T2DM model rats, with 14 components common to both groups. These shared components mainly included organic acids, terpenoids, glycosides, amino acids, and their derivatives. The two differential components in normal rats were identified as coumarin derivatives, while the seven differential components in T2DM model rats primarily consisted of flavonoids, terpenoids, and glycosides. Network pharmacology analysis identified 2191 diabetes-related targets, with degree-based screening yielding 32 key targets under physiological conditions and 46 under pathological conditions. GO and KEGG analyses suggested that the PI3K/AKT signaling pathway may play a key role in alleviating T2DM, and the prediction was subsequently validated in the T2DM rat. Molecular docking and MD studies revealed that Formononetin and Licorice saponin K2 exhibited strong binding affinity with AKT1, which was further confirmed by SPR results demonstrating effective interaction between these components and the target.

CONCLUSION: Component analysis results indicated that the in vitro constituents of SMYAD were primarily flavonoids, organic acids, amino acids, and terpenoids. Serum pharmacochemistry findings revealed differences in the types of serum migrant components between normal rats and T2DM model rats. Under pathological conditions, the differential components were predominantly flavonoids, terpenoids, and glycosides. This suggests that the pathological state influences the absorption of these components, and that they may be the potential active components responsible for alleviating T2DM. Network pharmacology analysis combined with animal experiments verified that the PI3K/AKT pathway is the key signaling mechanism for ameliorating insulin resistance in T2DM.