PB2 alleviates diabetic nephropathy: Intervene gut-flora-dependent metabolite TMAO promote DN-associated inflammatory responses through ferroptosis, ER stress–autophagy axis, and macrophage polarization

Diabetic nephropathy (DN), a condition characterized by persistent inflammatory responses, represents one of the most serious complications of diabetes mellitus; however, effective treatment strategies for DN remain limited. This study aimed to explore the potential therapeutic effects of procyanidin B2 (PB2) in mice with DN induced by high-fat diet (HFD) and streptozotocin (STZ). Tissue pathology was evaluated by histopathological analysis. Microbiome sequencing, targeted metabolomics, quantitative real-time polymerase chain reaction (qRT‒PCR), western blotting (WB), and immunohistochemistry (IHC) were combined to explore alterations in gut-derived trimethylamine N -oxide (TMAO) and the underlying mechanisms through which PB2 in prevents DN. Finally, TMAO rescue experiment and fecal microbiota transplantation (FMT) was performed to validate the regulatory effects of TMAO and the gut microbiota on DN. The results indicated that PB2 significantly improved liver and kidney function and intestinal-barrier integrity via the gut–liver–kidney axis while maintaining cholesterol homeostasis and restoring bile acid (BA) metabolism. Notably, PB2 significantly modulated the gut microbiota and markedly decreased circulating TMAO levels by targeting the trimethylamine/flavin-containing monooxygenase 3/TMAO axis. TMAO promoted renal injury by multiple signaling pathways, thereby inducing ferroptosis, stress–autophagy axis, macrophage polarization. Furthermore, TMAO supplementation and FMT verified the importance of the gut-flora-dependent metabolite TMAO in the protective role of PB2 against DN. Collectively, these findings clarify the anti-DN effects of PB2, which occur through a novel gut–liver–kidney axis-based mechanism, and suggest that PB2 is a promising therapeutic option for DN through the modulation of kidney inflammation.