Suppression of ferroptosis through the SLC7A11/glutathione/glutathione peroxidase 4 axis contributes to the therapeutic action of the Tangshenning formula on diabetic renal tubular injury.

backgroundTangshenning (TSN) is a safe and effective formula to treat diabetic nephropathy (DN), and clinical studies have demonstrated that its therapeutic effects are related to oxidative stress improvements in patients. Herein, this study aims to explore the potential mechanism of how TSN alleviates diabetic renal tubular injury.

methodsThe ultrahigh pressure liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-QTOF/MS) was used to identify the chemical composition and serum components of TSN. KK-Ay mice served to investigate the protective effects and regulatory mechanisms of TSN on tubular damage in DN. Furthermore, inhibitors and inducers of ferroptosis were employed in high glucose-cultured tubular epithelial cells (TECs) to verify the potential mechanisms of TSN. The expressions of proteins related to renal tubular injury, ferroptosis and solute carrier family 7, member 11 (SLC7A11)/glutathione (GSH)/glutathione peroxidase 4 (GPX4) axis were analyzed by western blot and immunofluorescence. Mitochondrial ultrastructure was observed in kidney tissues and TECs by a transmission electron microscope. Pathological changes in the renal tissues were observed by HE, PAS, and Prussian blue staining. Ferroptosis-related reactive oxygen species (ROS), malondialdehyde (MDA), ferrous ion, the intake of cystine, GSH, and oxidized glutathione (GSSG) were evaluated and contrasted in vivo or in vitro.

results51 compounds of TSN powder and 11 components in TSN-containing serum were identified by UPLC-QTOF/MS method. Administration of TSN ameliorated the elevated levels of proteinuria, serum creatinine, blood urea nitrogen, abnormal expression of renal tubular injury markers, and pathological damage to the renal tubules in DN mice model. Intriguingly, a strong inhibition of ferroptosis after TSN treatment occurred in both DN mice model and high glucose-cultured TECs. Notably, induction of ferroptosis by erastin attenuated the protective effect of TSN in high glucose-cultured TECs, while the ferroptosis inhibition by ferrostatin-1 treatment protected renal tubular, which was similar to TSN, suggesting the contribution of TSN-mediated by the inhibition of ferroptosis in DN progression. Mechanistically, TSN upregulated the SLC7A11/GSH/GPX4 axis to inhibit ferroptosis.

conclusionTSN may delay the DN progression and attenuate the renal tubular injury by inhibiting the ferroptosis regulated by the SLC7A11/GSH/GPX4 axis.