Vaccarin Ameliorates Renal Fibrosis by Inhibiting Ferroptosis via Nrf2/SLC7A11/GPX4 Signaling Pathway

Mengjiao Cui,1,2,* Qiming Xu,1,2,* Lianxiang Duan,1 Jianrao Lu,1 Jing Hu1 1Department of Nephropathy, The Seventh People’s Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200137, People’s Republic of China; 2Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jing Hu; Jianrao Lu, Department of Nephropathy, The Seventh People’s Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China, Email [email protected]; [email protected]: Vaccarin is a natural flavonoid glycoside with anti-inflammatory, antioxidant and nephroprotective effects. However, the effects of vaccarin on renal fibrosis (RF) and its molecular mechanisms remain unclear. This study aimed to investigate the effects of vaccarin on RF and its molecular mechanisms.Methods: Network pharmacology was used to analyze the effect of vaccarin on RF, and molecular docking and molecular dynamics simulations were performed to assess the binding of nuclear factor erythroid 2-related factor 2 (Nrf2) to vaccarin. A mouse model of unilateral ureteral obstruction (UUO) was established in vivo, and human renal tubular epithelial (HK2) cells were induced with transforming growth factor-β (TGF-β) and RSL3, respectively, as an in vitro model. The anti-fibrotic effect of vaccarin was observed by histopathological staining and determination of fibrous markers. Changes in oxidative stress and ferroptosis-related markers were detected by kits, Western blot (WB), qRT-PCR and immunofluorescence (IF). Finally, Nrf2 inhibitors were added to the in vitro model to observe the effects on fibrosis and ferroptosis.Results: Vaccarin and RF cross genes are enriched for oxidative stress. Nrf2 binds stably to vaccarin. Both in vivo and in vitro experiments showed that vaccarin treatment reduced the expression of fibrosis markers, decreased the levels of reactive oxygen species (ROS), malondialdehyde (MDA), lipid peroxidation (LPO) and Fe2+, and increased glutathione (GSH) secretion. In addition, vaccarin down-regulated the expression of Long-chain acyl-CoA synthetase 4 (ACSL4), prostaglandin-endoperoxide synthase 2 (PTGS2) and NADPH oxidase 1 (NOX1), and up-regulated Nrf2 and its downstream solute transport family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) expression. Mechanistic studies indicated that vaccarin activated the Nrf2/SLC7A11/GPX4 pathway to inhibit ferroptosis, and this inhibition was effectively reversed by the Nrf2 inhibitor.Conclusion: Vaccarin ameliorates RF by inhibiting ferroptosis via Nrf2/SLC7A11/GPX4 pathway.Keywords: vaccarin, renal fibrosis, Nrf2/SLC7A11/GPX4 pathway, ferroptosis, oxidative stress, network pharmacology