Targeting the NF-κB p65-MMP28 axis: Wogonoside as a novel therapeutic agent for attenuating podocyte injury in diabetic nephropathy.

Background: Although recent progress provides mechanistic insights into diabetic nephropathy (DN), effective treatments remain scarce. DN, characterized by proteinuria and a progressive decline in renal function, primarily arises from podocyte injury, which impairs the glomerular filtration barrier. Wogonoside, a bioactive compound from the traditional Chinese herb Scutellaria baicalensis, has not been explored for its role in DN.

Purpose: This study aimed to investigate the therapeutic effects of wogonoside on podocyte injury in DN and its molecular mechanisms.

Methods: The effects of wogonoside were examined using HFD/STZ-induced DN mouse models and high glucose (HG)-induced MPC-5 cells. Oxidative stress and inflammation markers were analyzed via Western blot and RT-qPCR. Wogonoside targets were identified through DARTS-MS and validated by SPR, molecular docking, alanine scanning, and CETSA. RNA-Seq analysis was employed to identify downstream targets, and the p65-MMP28 axis was explored through p65 knockdown and overexpression studies. The regulatory effect of p65 on Mmp28 was confirmed through dual-luciferase reporter assays and ChIP-qPCR.

Results: Wogonoside treatment significantly reduced oxidative stress and inflammation in vivo and in vitro. Mechanistic studies identified p65 as a direct target of wogonoside, with SPR confirming a strong binding affinity (K = 25.05 μM). Molecular docking and alanine scanning identified LYS221 as a critical binding site, which was further supported by CETSA using the p65 K221A mutant. RNA-Seq analysis revealed Mmp28 as a downstream effector of p65 involved in HG-induced podocyte injury. Functional studies demonstrated that wogonoside's protective effects on antioxidant and inflammatory pathways are mediated via the p65-MMP28 axis. Dual-luciferase reporter assays revealed that p65 regulates Mmp28 transcription, and ChIP-qPCR confirmed its direct promoter binding.

Conclusions: This study highlights wogonoside as a promising candidate for the treatment of podocyte injury in DN by targeting the NF-κB p65-MMP28 signaling axis. These findings provide novel insights into wogonoside's therapeutic potential and its molecular mechanisms, paving the way for its further development as a DN intervention.