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The pathogenesis of atherosclerosis is closely related to endothelial cell injury caused by lipid peroxidation-induced ferroptosis. Tanshinone IIA (TSA) protects endothelial tissues from damage. In this study, we investigated whether TSA exerts its protective effect on endothelial cells by inhibiting ferroptosis. Ferroptosis was induced in human coronary artery endothelial cells (HCAECs), and cells were treated with TSA. Morphological examination indicated that TSA exerted a significant protective effect on the HCAECs. This was further confirmed by LDH release and cell death detection assays. Flow cytometry revealed that TSA significantly reduced the excessive accumulation of total cellular ROS and lipid ROS caused by ferroptosis inducers. TSA also restored the reduction of glutathione (GSH), a potent and abundant reductant in cells. In addition, we found that TSA promoted the expression of NRF2, an essential player in response to oxidative stress, and its downstream genes. Immunofluorescent staining revealed that TSA promoted the nuclear translocation of NRF2. Increased nuclear translocation of NRF2 was validated by Western blot evaluation of cytoplasmic and nuclear protein extracts. Furthermore, NRF2 inhibition abolished the protective effects of TSA on HCAECs. These data demonstrate that TSA represses ferroptosis via activation of NRF2 in HCAECs.

MICALs are large, multidomain flavin-dependent monooxygenases that use redox chemistry to cause actin to depolymerize. Little enzymology has been reported for MICALs, and none has been reported for MICAL-2, an enzyme vital for the proliferation of prostate cancer. The monooxygenase domains of MICALs resemble aromatic hydroxylases, but their substrate is the sulfur of a methionine of actin. In order to determine how closely MICAL-2 conforms to the aromatic hydroxylase paradigm, we studied its reaction with NAD(P)H. The enzyme has a strong preference for NADPH over NADH caused by a large difference in binding NADPH. A comparison of the reduction kinetics using protio-NADPH and [4R-(2)H]-NADPH showed that MICAL-2 is specific for the proR hydride of NADPH, as evidenced by a 4.8-fold kinetic isotope effect. The reductive half-reaction of the MICAL-2 hydroxylase domain is stimulated by f-actin. In the absence of actin, NADPH reduces the flavin relatively slowly; actin speeds that reaction significantly. The separate monooxygenase domain of MICAL-2 has the classic regulatory behavior of flavin-dependent aromatic hydroxylases (Class A monooxygenases): slow reduction of the flavin when the substrate to be oxygenated is absent. This prevents the wasteful consumption of reduced pyridine nucleotide and the production of harmful H2O2. Our results show that this strategy is used by MICAL-2. Thus, our data suggest that MICAL-2 could regulate catalysis through the monooxygenase domain alone; control by interactions with other domains of MICAL in the full-length enzyme may not be needed.