Baicalein mitigates iron overload-induced cardiomyocyte injury via activating the Keap1/Nrf2/HO-1 pathway

LONG Xingjiang; QIN Jiaofeng; HUANG Xiangui

doi:10.3969/j.issn.1674-3806.2026.01.14

Abstract：Objective To explore the protective mechanism of baicalein(BAL)on cardiomyocyte injury caused by iron overload.Methods An iron-overload cell model was constructed by intervening cardiomyocyte H9c2 with 200 μmol/L ferric citrate(FC)for 24 hours.The protective effects of different concentrations of BAL(3,10,and 30 μmol/L)on cardiomyocytes with iron overload were analyzed.The protective effects of BAL on cardiomyocytes by inhibiting the Kelch-like ECH-associated protein1(Keap1)/nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)pathway(treated with HO-1 inhibitor SnPP or Nrf2 inhibitor ML385)were analyzed.Cell viability was detected by using the CCK-8 assay.The expression levels of inflammatory factors including tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)were detected by using real-time quantitative polymerase chain reaction(RT-qPCR).The level of reactive oxygen species(ROS)was detected by using 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA)fluorescent probe.The expression of 4-hydroxynonenal(4-HNE)was detected by using immunofluorescence staining.The expression levels of oxidative stress indicators of malondialdehyde(MDA)and glutathione(GSH),and iron content were detected by using reagent kits.The expression levels of proteins related to the Keap1/Nrf2/HO-1 pathway and ferroptosis were detected by using Western blot assay.Results BAL could enhance the viability of iron-overloaded H9c2 cells,reduce the expression levels of inflammatory factors of TNF-α,IL-1β and IL-6,as well as oxidative stress indicators of ROS and 4-HNE,making the expression level of MDA decrease and the expression level of GSH increase,and the intervention effects were dose-dependent.In addition,after intervention with BAL,iron deposition in the iron-overloaded H9c2 cells decreased,while the expressions of ferroptosia-related protein solute carrier family 7 member 11(SLC7A11),glutathione peroxidase 4(GPx4),and solute carrier family 3 member 2(SLC3A2)protein increased,and the expression of cyclooxygenase-2(COX-2)protein decreased.The results of Western blot assay showed that BAL activated the Keap1/Nrf2/HO-1 pathway,inhibited the expression of Keap1 protein,and promoted the expressions of Nrf2 and HO-1 proteins,and the intervention effects were dose-dependent.After treatment with SnPP or ML385,the protective effect of BAL on iron-overloaded H9c2 cells weakened,and the cell viability decreased,and the expressions of inflammatory factors increased,and the oxidative stress and lipid peroxidation enhanced,and the iron deposition increased,and the ferroptosis intensified.Conclusion BAL may mitigate iron overload-induced cardiomyocyte injury via activating the Keap1/Nrf2/HO-1 pathway.