Neferine, a major alkaloid from the traditional Chinese medicine Lian Zi Xin, alleviates renal fibrosis by targeting GSK3β and β-catenin signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Neferine (Nef) is the primary bioactive alkaloid of lotus seed germ (Lian Zi Xin), a traditional Chinese medicine long used for 'clearing heart fire' and ' eliminating internal heat, and regulating body fluid metabolism (e.g., promoting diuresis and reducing edema)'. From the perspective of modern medical concepts, 'internal heat' and 'fluid imbalance' often correspond to conditions such as impaired kidney function. AIM OF THE STUDY: To investigate the protective effects of Nef against renal injury in mice and identify the underlying mechanisms. MATERIALS AND METHODS: In vitro model of diabetic nephropathy (DN) and renal fibrosis were established using high glucose-stimulated mesangial cells and TGF-β1-induced HK-2 cells, respectively. In vivo model of DN and renal fibrosis were established by high-fat diet combined with streptozotocin, and unilateral ureteral obstruction (UUO) , respectively. Nef intervention was applied in both models. Fibrotic markers such as fibronectin (FN), collagen IV (Col IV) and key components of the Wnt/β-catenin pathway were assessed by Western blot and immunohistochemistry. The binding between neferine and target proteins was validated through bioinformatics, pull-down assays, and cellular thermal shift assay (CETSA). The regulatory effects of Nef on GSK3β phosphorylation (Ser9) and β-catenin accumulation were further examined. RESULTS: Treatment with neferine results in significant downregulation of extracellular matrix components, such as FN and Col IV, along with inhibition of epithelial-mesenchymal transition in both cellular and animal models. At the molecular level, Nef exerts its effects by directly binding to β-catenin and GSK3β, reducing GSK3β phosphorylation at Ser9, which in turn prevents β-catenin accumulation and suppresses Wnt/β-catenin pathway activity. CONCLUSION: Nef ameliorates DN and UUO induced renal injury by targeting GSK3β and β-catenin, thereby inhibiting the Wnt/β-catenin signaling pathway. These findings highlight its therapeutic potential for fibrotic kidney diseases.