Yohimbine attenuates oxidative stress through circGNB1/CDA1 axis in diabetic nephropathy

Abstract

Aim: To investigate the role of yohimbine (YH) in diabetic nephropathy (DN) progression and identify its underlying molecular mechanisms.

Methods: Human renal proximal tubular epithelial cells (HK-2) were cultured with high glucose (HG) and treated with varying doses of YH. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Inflammatory markers and oxidative stress were measured. Male diabetic C57BLKS/J-LepR (db/db) mice received YH (2.5, 5, or 10 mg/kg) daily for eight weeks. Circular RNA (circRNA) microarray analysis was performed on HG-induced HK-2 cells. RNA pull-down assays combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify circGNB1-interacting proteins. Cellular localization and protein expression were determined through fractionation, RNA fluorescence in situ hybridization (RNA-FISH), and western blotting.

Results: YH significantly enhanced viability of HG-induced HK-2 cells while reducing inflammatory factors and oxidative stress markers. These protective effects were confirmed in db/db mice, where YH reduced blood glucose, urinary albumin-to-creatinine ratio, and blood urea nitrogen levels. CircRNA microarray identified circGNB1 as the most significantly upregulated gene in YH-treated HG-induced cells. RNA pull-down assays identified cell division autoantigen 1 (CDA1) as a circGNB1-interacting protein. CircGNB1 positively regulated CDA1 expression at the protein level in HK-2 cells. CDA1 knockdown significantly decreased cell viability and counteracted the protective effects of YH treatment.

Conclusion: Our study identifies a novel YH/circGNB1/CDA1 axis in DN progression. YH attenuates oxidative stress through upregulation of circGNB1, which interacts with and regulates CDA1 protein expression, thereby mitigating HG-induced renal cell damage. These findings provide new insights for developing therapeutic strategies for DN.

Keywords

Diabetic nephropathy / yohimbine / circGNB1 / CDA1 / oxidative stress / high glucose / renal protection

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