Effects Of Peroxidation And GPx4 Expression On Pulmonary Fibrosis And Ferroptosis

Objective To explore the relationship of ferroptosis in the occurrence and development of human pulmonary fibrosis,and to study the effect of lipid peroxidation which closely linked with ferroptosis on pulmonary fibrosis through the expression of GPx4.Methods The human lung fibroblasts cells(HFL1)cultured in vitro were divided into five groups,namely,in the blank control group,in the TGF-?1 group,the TGF-?1+erastin group,the TGF-?1+Fer-1 group,the TGF-?1+erastin+Fer-1 group,and the respective drug concentration was TGF-?1 6ng/ml,the erastin 2.5?M,and the Fer-11?M.After 24 hours of cell starvation treatment,we treated cells at 6,12,24,36 hours.The specific methods are as follows:observation of cell growth status by optical microscope and observation of intracellular ultrastructure by electron microscope;Cell survival rate was detected by CCK-8.The contents of reactive oxygen species(ROS)and malondialdehyde(MDA)in cells were determined by fluorescence spectrophotometry;The ?-SMA,COL I and GPx4 mRNA expression in each group was detected by real-time quantitative PCR,and the protein expression of GPx4 in each group was detected by means of immunofluorescence.Results Cell death appeared at 12h and gradually aggravated after treatment with TGF-?1 and erastin.TEM analysis showing the mitochondria of HFL1 cells in TGF-?1-treated group were smaller,with fewer of mitochondria crista.The mitochondria in HFL1 cells of TGF-?1+erastin treated group were smallest,with fewest/vanishing of mitochondria crista at 3 6h.Compared with control group and TGF-?1 group,the cell viability rate was obviously decreased in TGF-?1+erastin treated group from 12h,and further reduced with the time until 36h(P<0.01).The levels of ROS,MDA were increased after treatment with TGF-?1,and all these were magnified after TGF-?1+erastin treatment.All these changes induced by TGF-?1 and erastin can be recovered by Fer-1 treatment.The ?-SMA and COL I mRNA expression levels increased significantly from 24h after TGF-?1-treatment,and further rose after TGF-?1+erastin treatment.the levels of GPX4 mRNA and protein were reduced after treatment with TGF-?1,and all these were magnified after TGF-?1+erastin treatment.All these changes induced by TGF-?1 and erastin can be recovered by Fer-1 treatment.Conclusion Our study showed that GPx4 is inhibited in myofibroblast differentiation and ferroptosis,which leads to the overproduction of lipid peroxides.increasing of ROS and lipid peroxidation,inhibition of GPX4 were common causes for pulmonary fibrosis and ferroptosis induced by erastin,but not sufficient conditions for ferroptosis.Fer-1 can improve the GPX4 expression and reduce lipid peroxidation,resulting in suppression of fibroblast-to-myofibroblast differentiation.