Department of Endocrinology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029 ,China
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Abstract:
Objective:To explore whether atorvastatin(Ator)can induce ferroptosis in pancreatic β- cell line MIN6 cells and its possible mechanism. Methods:MIN6 cells were divided into control group,Ator group,Ator+apoptosis inhibitor(Z-VAD-FMK)group, Ator+necrostatin-1(Nec-1)group and Ator+ferrostatin-1(Fer-1)group. Cell viability was detected by cell counting kit-8(CCK-8) method. The ultrastructure of cells was observed by transmission electron microscopy. The levels of reactive oxygen species(ROS)and Fe2+ were observed by fluorescence microscopy. The contents of malondialdehyde(MDA)and glutathione(GSH)were detected by enzyme-linked immunosorbent assay(ELISA)method. Real-time quantitative PCR was used to detect the mRNA levels of caspase-3, receptor-interacting protein kinase 3(Ripk3),acyl-CoA synthetase long-chain family member 4(Acsl4),prostaglandin endoperoxidase synthase 2(Ptgs2)and glutathione peroxidase 4(Gpx4). Western blot was used to detect the proteins levels of 4-hydroxynonenal (4-HNE)and GPX4. Results:Compared with the Ator group,cell viability of MIN6 was higher in Ator+Z-VAD-FMK group and Ator+ Fer -1 group(P < 0.01). MIN6 cells,which were treated with Ator,exhibited the characteristic morphologic features associated with apoptosis,ferroptosis and autophagy under transmission electron microscopy. Compared with the control group,the levels of the intracellular Fe2+ ,MDA and ROS in the Ator group were increased and GSH was decreased. The mRNA relative expression levels of caspase-3,Acsl4 and Ptgs2 were increased,as well as the protein relative expression level of 4-HNE(all P < 0.05). The mRNA and protein relative expression levels of GPX4 were decreased(P < 0.05). Compared with the Ator group,the levels of the intracellular Fe2+ ,MDA and ROS in the Ator+Fer-1 group were decreased and GSH was increased. The mRNA relative expression level of Acsl4 was decreased and Gpx4 was increased(all P < 0.05). The protein relative expression levels of 4-HNE was decreased and GPX4 was increased,though the changes were not statistically significant. Conclusion:Atorvastatin may induce ferroptosis in MIN6 cells by down-regulating GPX4 expression through inhibiting mevalonate pathway.