Rhoifolin Attenuates Ferroptosis in Renal Tubular Epithelial Cells in Diabetic Nephropathy by Targeting CDKN1A to Modulate the CDK2/E2F1 Pathway

Background: Current therapeutic strategies for Diabetic nephropathy (DN) remain limited, underscoring the urgent need to identify novel pharmacological agents and therapeutic targets. Methods: Network pharmacology analysis was conducted to identify key genes as principal target genes of Rhoifolin (ROF) in treating DN. A mouse model of DN was established using streptozotocin induction combined with a high-fat diet. Mice were randomly allocated into six groups: Control, DN, DN+ROF-low (L), DN+ROF-medium (M), DN+ROF-high (H), and DN+metformin (MET). In vitro, a high glucose (HG)-induced injury model was established in HK-2 cells. Cells were assigned to the normal control (NC), HG, HG+ROF-L, HG+ROF-M, HG+ROF-H, HG+ROF-H+overexpression negative control (oe-NC), HG+ROF-H+oe-cyclin dependent kinase inhibitor 1A (CDKN1A), HG+ROF-H+oe-CDKN1A+oe-NC, HG+ROF-H+oe-CDKN1A+oe-cyclin-dependent kinase 2 (CDK2), and HG+ROF-H+oe-CDKN1A+ferrostatin-1 (Fer-1) groups. Fasting blood glucose (FBG), body weight, and urinary albumin-to-creatinine ratio were measured; histopathological alterations were evaluated using periodic acid-schiff and hematoxylin-eosin staining; glutathione and malondialdehyde levels were quantified using corresponding detection kits; reactive oxygen species levels were assessed using dihydroethidium fluorescent probes; and the protein expression levels of CDKN1A, glutathione peroxidase 4 (GPX4), Acyl-CoA synthetase long chain family member 4 (ACSL4), CDK2, E2F transcription factor 1 (E2F1) were analyzed by western blot. Cell viability was determined using CCK-8; cell death was quantified by flow cytometry; levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) were determined using enzyme-linked immunosorbent assay; and Fe2+ concentrations were measured with fluorescent probes. Results: ROF treatment significantly alleviated renal injury in mice with DN, enhanced the viability of HK-2 cells, activated the CDK2/E2F1 pathway, and inhibited ferroptosis. Overexpression of CDKN1A in the HG+ROF-H group resulted in decreased cell viability, increased cell death, elevated secretion of TNF-α, IL-1β, IL-6, enhanced ferroptosis, and suppression of the CDK2/E2F1 pathway. Administration of the ferroptosis inhibitor Fer-1 in the HG+ROF-H+oe-CDKN1A group attenuated CDKN1A-induced cellular injury. Furthermore, overexpression of CDK2 in the HG+ROF-H+oe-CDKN1A group inhibited ferroptosis and mitigated the cellular injury caused by CDKN1A overexpression. Conclusion: ROF mitigates the progression of DN by targeting CDKN1A and modulating CDK2/E2F1 pathway-mediated ferroptosis in HK-2 cells.