Puerarin Regulates The Nrf2/SLC7A11/GPX4 Pathway To Inhibit H₂O₂-induced RSC96 Cell Ferroptosis

Objective:To examine the protective effect of puerarin on H₂O₂-induced ferroptosis in RSC96 cells and to elucidate the underlying mechanisms.Methods:In vitro cultured RSC96 cells were randomly assigned into four groups:the control group（Control,Con）,model group（H₂O₂）,puerarin pretreatment group（H₂O₂+Pue）and Nrf2 inhibitor ML385 pretreatment group（H₂O₂+Pue+ML385）.The following experiments were subsequently conducted:1.In order to determine the concentration and duration of H₂O₂treatment for ferroptosis induction in RSC96 cells,the cells were treated with H₂O₂at a concentration range of 0～0.75 m M,and Western blot（WB）was performed to measure the protein expression level of GPX4 and PTGS2 proteins related molecules.2.In order to determine the concentration of puerarin intervention0.25～1 m M puerarin pretreatment was administered to RSC96 cells,add 0.3 m M concentration of H₂O₂ to RSC96 cells for 24 h,CCK-8 to examine cell viability.3.WB was performed to analyze the SLC7A11,GPX4,Nrf2 and HO-1 protein levels in the cells.4.GSH,MDA,T-SOD,ROS and Fe²⁺levels were analyzed using kit.5.Immunofluorescence assay was performed for Nrf2 colocalization analysis.Results:1.Compared with the Con group,RSC96 cells treated with 0.3 m M H₂O₂ for 24 h showed significantly decreased GPX4 and drastically increased PTGS2 protein expression level（P<0.05 or P<0.01）,indicating induced ferroptosis in RSC96 cells.2.Compared with the Con group,the cell viability of the H₂O₂ group was significantly reduced（P<0.01）;Compared with the H₂O₂ group,RSC96 cells pretreated with 500μM puerarin for 30 minutes showed a significantly higher cell survival rate（P<0.01）.3.The expression of GPX4,SLC7A11,HO-1 and Nrf2 proteins in the puerarin retreated group was significantly increased（P<0.05 or P<0.01）.4.Compared with the Con group,the levels of endogenous antioxidants represented by RSC96 intracellular glutathione（GSH）and superoxide dismutase（SOD）in the H₂O₂ group decreased,and the accumulation of reactive oxygen species（ROS）and malondialdehyde（MDA）,the end product of lipid peroxidation,increased（P<0.05 or P<0.01）;Compared with the H₂O₂ group,the levels of intracellular GSH and SOD in the puerarin retreated group were significantly increased,and the accumulation of ROS and MDA decreased significantly.5.Immunofluorescence showed that compared with H₂O₂ group,the colocalization coefficient of H₂O₂+Pue group was significantly increased（P<0.05）,and this effect was inhibited by the inhibitor ML385 of Nrf2.Conclusion:Puerarin has a significant protective effect against H₂O₂-induced ferroptosis in RSC96 cells within a certain dose range,and the potential mechanisms include activation of the Nrf2/SLC7A11/GPX4 signaling pathway and inhibition of lipid peroxidation.