Li Xiandeng, Zhao Shuyan, Xie Jing, Li Mi, Tong Shuangmei, Ma Jing, Yang Rui, Zhao Qinjian, Zhang Jian, Xu Ajing
Department of Clinical Pharmacy, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China; College of Pharmacy, Chongqing Medical University, Chongqing, 400016, China.
Department of Clinical Pharmacy, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China.
Phytomedicine. 2025 Mar;138:156406. doi: 10.1016/j.phymed.2025.156406. Epub 2025 Jan 24.
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.
This study aimed to investigate the therapeutic effects of wogonoside on podocyte injury in DN and its molecular mechanisms.
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.
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.
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.
尽管最近的进展为糖尿病肾病（DN）提供了机制上的见解，但有效的治疗方法仍然稀缺。DN以蛋白尿和肾功能进行性下降为特征，主要源于足细胞损伤，这会损害肾小球滤过屏障。汉黄芩苷是一种来自传统中药黄芩的生物活性化合物，其在DN中的作用尚未得到研究。
本研究旨在探讨汉黄芩苷对DN中足细胞损伤的治疗作用及其分子机制。
使用高脂饮食/链脲佐菌素诱导的DN小鼠模型和高糖（HG）诱导的MPC-5细胞来检测汉黄芩苷的作用。通过蛋白质印迹法和逆转录定量聚合酶链反应（RT-qPCR）分析氧化应激和炎症标志物。通过药物亲和反应靶标稳定性质谱法（DARTS-MS）鉴定汉黄芩苷的靶点，并通过表面等离子体共振（SPR）、分子对接、丙氨酸扫描和细胞热位移分析（CETSA）进行验证。采用RNA测序（RNA-Seq）分析来鉴定下游靶点，并通过p65基因敲低和过表达研究探索p65-MMP28轴。通过双荧光素酶报告基因测定和染色质免疫沉淀定量聚合酶链反应（ChIP-qPCR）证实p65对Mmp28的调控作用。
汉黄芩苷治疗显著降低了体内和体外的氧化应激和炎症。机制研究确定p65是汉黄芩苷的直接靶点，SPR证实其具有很强的结合亲和力（K = 25.05 μM）。分子对接和丙氨酸扫描确定赖氨酸221（LYS221）为关键结合位点，使用p65 K221A突变体的CETSA进一步支持了这一点。RNA-Seq分析显示Mmp28是参与HG诱导的足细胞损伤的p65下游效应分子。功能研究表明，汉黄芩苷对抗氧化和炎症途径的保护作用是通过p65-MMP28轴介导的。双荧光素酶报告基因测定显示p65调节Mmp28转录，ChIP-qPCR证实其直接与启动子结合。
本研究强调汉黄芩苷作为一种有前景的候选药物，可通过靶向核因子κB p65-MMP28信号轴治疗DN中的足细胞损伤。这些发现为汉黄芩苷的治疗潜力及其分子机制提供了新的见解，为其作为DN干预措施的进一步开发铺平了道路。
