Mechanistic study on the male flower of Eucommia ulmoides-Sanchi flower herb pair for improving glucose and lipid metabolism in type 2 diabetes mellitus model mice

Based on the traditional Chinese medicine theory of "simultaneous regulation of the liver and kidney," this study integrated network pharmacology prediction, molecular docking, and animal experimental validation to analyze the multi-target regulatory network of Duzhong Xionghua (the male flower of Eucommia ulmoides)-Sanqi Hua (Sanchi flower) herb pair (hereinafter called "herb pair") in modulating glucolipid metabolic disorders in type 2 diabetes mellitus (T2DM), thereby elucidating its underlying molecular mechanism.

Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, China National Knowledge Infrastructure, VIP Database for Chinese Technical Periodicals, and Wanfang Data were used to obtain the active ingredients of the male flower of Eucommia ulmoides and Sanchi flower. PubChem Compound, SwissTargetPrediction, and SuperPred were used to screen and predict the targets of the drugs; The Human Gene Database, The Online Mendelian Inheritance in Man, and Therapeutic Target Database were used to screen the key gene targets of T2DM. A "component-target-pathway" network diagram was constructed using Cytoscape software, and Gene Ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were employed to identify the functions of the relevant target genes and pathways. Molecular docking was used to verify the binding activities of the core components and the key targets. For animal experiments, spontaneous T2DM model mice were used, in which the normal group consisted of six mice (wild type) from the same litter, and the 24 successfully modeled mice were randomly divided into model, metformin (0.26 g/kg), high-dose herb pair (2.6 g/kg), and low-dose herb pair groups (1.3 g/kg) according to the blood glucose levels and body weights, with six mice per group. The drugs were administered by gavage daily for six consecutive weeks. The body weight and fasting blood glucose (FBG) levels were measured weekly, and an oral glucose tolerance test was performed in the fifth week. At the end of drug administration, body weight, naso-anal length, liver and bilateral epididymal adipose mass were measured; pathological changes in the liver were observed using HE staining; serum levels of aspartate transaminase (AST), alanine amino-transferase (ALT), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected using colorimetric assay; and liver tissue phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/glycogen synthase kinase-3β (GSK-3β) signaling pathway protein expressions were determined using Western blotting.

Network pharmacology screening identified 38 active components and 669 potential targets of the herb pair. Intersection analysis with 1, 275 T2DM-related targets yielded 185 common targets. Protein-protein interaction network analysis and pathway enrichment revealed the PI3K/AKT signaling pathway as a key mechanism. Molecular docking confirmed the strong binding affinity of the core components to key targets such as AKT1, suggesting that the herb pair may activate the PI3K/AKT pathway and inhibit GSK-3β activity via beta-sitosterol etc. Animal experiments demonstrated that, compared with the model group, the metformin group exhibited reduced FBG, AST, and ALT levels (P < 0.01), but failed to improve body weight, Lee′s index, or epididymal fat coefficient. Both herb pair doses significantly lowered Lee′s index, hepatic index, and the epididymal fat coefficient (P < 0.01), with the low-dose herb pair group showing attenuated body weight gain in mice. In contrast, the high-dose herb pair group exhibited decreased FBG, improved glucose tolerance, reduced TC, TG, and LDL-C levels, and increased HDL-C level (all P < 0.01). HE staining revealed that all metformin and the herb pair markedly restored hepatic structure and alleviated steatosis in model mice, with more pronounced effects in the high-dose group than in the low-dose group. Western blotting result indicated that in the low-dose herb pair group, phospho-PI3K (p-PI3K), AKT, and phospho-GSK-3β (p-GSK-3β) protein expressions significantly increased (P < 0.05 or P < 0.01), whereas GSK-3β decreased (P < 0.05). The high-dose group exhibited enhanced PI3K, p-PI3K, AKT, phospho-AKT, and p-GSK-3β protein expressions (all P < 0.01), accompanied by reduced GSK-3β expression (P < 0.01).

The male flower of Eucommia ulmoides-Sanchi flower herb pair may ameliorate T2DM-related glucolipid metabolic disorders by modulating the PI3K/AKT/GSK-3β pathway.