Integrated network pharmacology and pharmacological investigations to discover the active compounds of Toona sinensis pericarps against diabetic nephropathy

Ethnopharmacological relevance: Toona sinensis (A. Juss.) Roem. Is a deciduous woody plant native to Eastern and Southeastern Asia. Different parts of this plant have a long history of being applied as traditional medicines to treat various diseases. The fruits have been used for antidiabetic, antidiabetic nephropathy (anti-DN), antioxidant, anti-inflammatory, and other activities. Aim of the study: The purpose of this study was to investigate the effects of EtOAc (PEAE) and n-BuOH extracts (PNBE) from T. sinensis pericarps (TSP) on kidney injury in high-fat and high-glucose diet (HFD)/streptozotocin (STZ)-induced DN mice by network pharmacology and pharmacological investigations, as well as to further discover active compounds that could ameliorate oxidative stress and inflammation, thereby delaying DN progression by regulating the Nrf2/NF-kappa B pathway in high glucose (HG)-induced glomerular mesangial cells (GMCs). Materials and methods: The targets of TSP 1-16 with DN were analyzed by network pharmacology. HFD/STZ-induced DN mouse models were established to evaluate the effects of PEAE and PNBE. Six groups were divided into normal, model, PEAE100, PEAE400, PNBE100, and PNBE400 groups. Fasting blood glucose (FBG) levels, organ indices, plasma MDA, SOD, TNF-alpha, and IL-6 levels, as well as renal tissue Nrf2, HO-1, NF-kappa B, TNF-alpha, and TGF-beta 1 levels were determined, along with hematoxylin-eosin (H&E) and immunohistochemical (IHC) analysis of kidney sections. Furthermore, GMC activity screening combined with molecular docking was utilized to discover active compounds targeting HO-1, TNF-alpha, and IL-6. Moreover, western blotting assays were performed to validate the mechanism of Nrf2 and NF-kappa B in HG-induced GMCs. Results: Network pharmacology predicted that the main targets of PEAE and PNBE in the treatment of DN include IL-6, INS, TNF, ALB, GAPDH, IL-1 beta, TP53, EGFR, and CASP3. Additionally, major pathways include AGE-RAGE and IL-17. In vivo experiments, treatment with PEAE and PNBE effectively reduced FBG levels and organ indices, while plasma MDA, SOD, TNF-alpha, and IL-6 levels, renal tissue Nrf2, HO-1, NF-kappa B, TNF-alpha, and TGF-beta 1 levels, and renal function were significantly improved. PEAE and PNBE significantly improved glomerular and tubule injury, and inhibited the development of DN by regulating the levels of oxidative stress and inflammation-related factors. In vitro experiments, compound 11 strongly activated HO-1 and inhibited TNF-alpha and IL-6. The molecular docking results revealed that compound 11 exhibited a high binding affinity towards the targets HO-1, TNF-alpha, and IL-6 (<-6 kcal/mol). Western blotting results showed compound 11 effectively regulated Nrf2 and NF-kappa B p65 protein levels, and significantly improved oxidative stress damage and inflammatory responses in HG-induced GMCs. Conclusion: PEAE, PNBE, and their compounds, especially compound 11, may have the potential to prevent and treat DN, and are promising natural nephroprotective agents.