Ferrostatin-1 Exerts Protective Effects On PQ-induced Acute Lung Injury By Repressing Ferroptosis

Objective: Although it is beneficial to improve agricultural production,Paraquat(PQ)itself is extremely high toxic to human.Once ingested,PQ rapidly produces a large number of oxidative stressors which destroy the structural integrity of cells and induce cell death quickly.Then,people who is bad condition will die due to respiratory failure.Interestingly,the pathogenesis of lung injury in paraquat poisoning has not been fully known.Ferroptosis is a novel form of programmed cell death that differs from traditional forms of cell death(such as necrosis,apoptosis and autophagy)and has been shown to play a role in many systemic diseases.Ferrostatin-1"Fer-1#is first inhibitor of Ferroptosis all of the word,which can reduce the incidence of Ferroptosis by inhibiting lipid peroxidation.At present,we can make a conclusion that there are still have another ways of cell death in PQ-induced ALI.So,we make a hypothesis that Ferroptosis may be involved in the pathogenesis of lung injury in paraquat poisoning.And,we also investigate the role of Fer-1 on PQ-induced ALI.METHODS: Male C57BL/6 mice and A549 cells were selected as experimental subjects for this experimental study.In the in vivo experiment,SPF-grade healthy male C57BL/6 mice were randomly divided into four groups(n=6),setting up the normal control(CON)group,paraquat poisoning(PQ)group,paraquat poisoning + Fer-1treatment(PQ+Fer-1)group and Fer-1 alone group,respectively.We first pretreated the mice by intraperitoneal injection of 2.5 μM/kg Fer-1 for 2 h.Then,we established an acute lung injury model using a dose of 30 mg/kg PQ,and anesthetized and collected lung tissue samples from mice at 72 h after poisoning.The severity of acute lung injury in each group was assessed by H&E staining and wet/dry weight ratio measurement.The levels of MDA and SOD in lung tissues were used to assess the degree of oxidative damage.SLC7A11 and GPX4 using Western-blot to investigate the expression levels of indicators of iron death and their role in PQ-induced acute lung injury.For in vitro studies,we used 400 μM PQ to stain A549 cells and divided them into CON,PQ,PQ+ Fer-1 and Fer-1 group.After 24 h of cell culturing,the pretreatment effect of 0.1μM Fer-1 drug was performed for 6 h before PQ was given for staining.The CCK-8 method was used to detect cell viability,and then cell supernatants and cells were collected and subjected to WB and PCR assays,as well as the detection of some phenotypic phenomena(such as the expression level of ROS,the content of MDA and the expression level of SOD).Results: HE staining results showed that PQ staining poisoned the alveoli of mice with structural destruction,cell wall thickening and hemorrhage.Prussian blue staining showed that the PQ group outlines blue stained areas compared to the CON group.The W/D ratio of lung tissue was also significantly increased in the PQ group compared with the CON group.m RNA levels of TNF-α,IL-1β and PTGS2 were significantly increased in the PQ group;secondly,MDA content was increased,SOD activity was decreased,and ROS levels were significantly increased.protein levels of TFR1 and ACSL4 were increased,and protein expression levels of SLC7A11 and GPX4 were decreased.With the use of Fer-1,the results all reverted and the lung injury in the mice was reduced.CONCLUSION: In vivo and ex vivo experiments,we can conclude that Fer-1 has protective effects against paraquat-induced acute lung injury by the inhibition of Ferroptosis.