PPARα Alleviates Iron Overload–induced Ferroptosis In Mouse Livers Through GPX4

Ferroptosis is a recently identified iron-dependent form of non-apoptotic cell death implicated in liver,brain,kidney,and heart pathology.Recent studies indicate that ferroptosis contributes to pathological process in a variety of diseases and conditions,including acute organ failure secondary to ischemia/reperfusion,huntington disease,and other neurodegenerative diseases.Thus,inhibiting ferroptosis may represent a promising new approach for treating cell death–related diseases.Ferroptosis can be induced by genetic deletion of the glutathione-dependent antioxidant enzyme GPX4.As an essential regulator of lipid peroxidation,GPX4 identified as the central regulator of ferroptosis,acting through the suppression of lipid peroxidation generation.Research has shown that GPX4 activator can decrease the intracellular ROS level and suppress ferroptosis.PPARαis a nuclear receptor that belongs to the steroid hormone receptor superfamily.PPARαstimulates the expression of target genes directly through binding to PPREs in the promoter regions of target genes.Therefore,we explored GW7647-activated PPARαwhether increases the expression of the GPX4 in the livers of mice,and alleviates ferroptosis caused by HID.We carried out the studies as follows:（1）Activation of PPARαsuppresses iron overload-induced ferroptosis in vivo.To investigate the role of PPARαin iron overload–induced ferroptosis,mice were fed a high-iron diet or injected dextriferron intraperitoneally and were orally gavage with either PPARαligand（GW7647）or vehicle.Iron administration strongly elevated the content of iron in liver,and as expected,GW7647 gavage significantly decreased the iron content.Lipid peroxidation and PTGS2 mRNA levels were significantly increased,and GSH content was decreased in mice fed with iron administration.Specific PPARαagonist GW7647 significantly reversed iron-induced the rising MDA content and PTGS2 mRNA levels,and the markly decreased GSH content.These data indicate that iron overload induced ferroptosis in vivo,and specific PPARαligands prevent this process upto some limit.（2）PPARαactivation induces GPX4 gene expression by binding to an IR-0 element in intron 3 of GPX4.Mice were administered by oral gavage with either vehicle or GW7647 twice a day for two days.Expression of GPX4 was significantly increased in mRNA and protein level after GW7647 treatment.The data indicated that PPARαpromoted GPX4expression.Hep1-6 cells were transfected with the pGL3-GPX4 reporter plasmid.Cells were subsequently treated with GW7647 for 24 h.Dual luciferase reporter gene assay showed that the transcriptional activity of pGL3-GPX4 reporter plasmid was significantly enhanced by GW7647 compared to the control group.ChIP assays demonstrate that PPARαbinds to the intronic enhancer in vivo.（3）A fluorescent probe was synthesized that recognizing behavior displays high selectivity towards Fe²⁺.To demonstrate the biologically relevant utility of the Fe²⁺specific fluorescent probe,culturing hep1-6 were incubated with 20μM probe in culture medium for 30min at 37℃,and very weak intracellular fluorescence inside the living cells was observed.Moreover,addition of external Fe²⁺produces enhanced intracellular fluorescence intensity,while limited increase intracellular fluorescence was observed with Cu²⁺and Fe³⁺at the same concentration.These results suggest that the highly cell permeable probe is able to facilely and specific respond to labile Fe²⁺iron pools.（4）PPARαdeficiency influence iron metabolism in the liver.To investigate the iron changes in metabolism,we used a HID diet eliciting substantial iron accumulation in the liver.A significant increase in intracellular fluorescence in the liver of PPARα^-/-mice compare to WT mice was observed.The hepatic iron content in PPARα^-/-mice increased significantly comparing with the WT group,indicating sever iron accumulation in the liver of PPARα^-/-mice.These results suggest that PPARαdeficiency induced significant increase in iron intake.（5）PPARαdeletion increases susceptibility to iron overload-induced ferroptosis.Feeding WT and PPARα^-/-mice the HID for 3 weeks or injecting dextriferron intraperitoneally for 2 weeks led to significantly higher tissue MDA content and PTGS2 mRNA level in PPARα^-/-mice compared with WT mice.We also found that hepatic GSH content in PPARα^-/-mice was lower than that of the WT mice after the HID.These results indicated that deficiency PPARαmarkedly increases iron overload-induced liver injury and the PPAR^-/-mice are more sensitive to ferroptosis.