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

Abstract

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_D = 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.