Mitoquinone Alleviates Bleomycin-induced Acute Lung Injury Via Inhibiting Mitochondrial ROS-dependent Pulmonary Epithelial Ferroptosis

Objective:the purpose of the present study was to explore the protective effects of pretreatment with MitoQ on bleomycin-induced ALI and lung epithelial ferroptosis.Method:Animal experiment:First,male C57BL/6J mice were intratracheally injected with bleomycin(BLM)(3.0 mg/kg)to establish an animal model of acute lung injury at different time,according to the preliminary dose of the research group.Male 8 weeks SPF C57BL/6J mice were randomly divided into two groups:control group and BLM group.Lung tissues of BLM group mice were collected at 6h、24h、72h times after exposure of BLM.Control group were intratracheally injected with same nomal saline.The levels of pulmonary,glutathione peroxidase 4(GPX4)、Ferroptosis suppressor protein-1(FSP1)and Tunel were detected.Subsequently,C57BL/6J mice were randomly divided into control group,Fer-1 group,BLM group and Fer-1+BLM group.Before intratracheally injected with BLM,mice were intraperitoneal injection Fer-1 1h in advance.Killing mice and obtaining lung tissue after 24h,the left lung was fixed in 4%paraformaldehyde and put in liquid nitrogen at once.In addition,the par of right lung tissue was collected by Transmission electron microscopy(TEM),the rest of which were detected malonaldehyde(MDA)and glutathione(GSH).Finally,C57BL/6J mice were randomly divided into control group,MitoQ group,BLM group and BLM+MitoQ group.Before intratracheally injected with BLM,mice were intraperitoneal injection MitiQ 1h in advance.After 24h,mice were sacrificed and the left lung was fixed in 4%paraformaldehyde and put in liquid nitrogen at once to HE staining.The par of right lung tissue was collected by Transmission electron microscopy(TEM),the rest of which were detected MDA and GSH.Cell experiment:First we ensured BLM dose on the basis of CCK-8 test.Subsequently,after 6h 12h and 24h of exposeing BLM(10 μg/mL),human bronchial epithelial cells(BEAS-2B)were collected for mitochondrial membrane potentials(MMPs)、oxidized lipids and mitochondrial reactive oxygen species(mtROS).BEAS-2B cells were then divided into control group,MitoQ group,BLM group and MitoQ+BLM group.MitoQ pretreatment was performed before BLM exposure,BEAS-2B were tested for MMPs、oxidized lipids、and mtROS after 24h.Results:Alveolar structure damage,followed by bleeding edema,inflammatory cell infiltration and pulmonary interstitial inflammation in mice is the major pathological feature for BLM-induced ALI.Oxidized lipids were upregulated in BLM-exposed BEAS-2B cells.Ferroptosis-characteristic ultrastructure,mainly disappearance of mitochondrial bilayer membrane structure and cristae,was observed in BLM-exposed pulmonary epithelium.Ferrostatin-1,a specific inhibitor of ferroptosis,attenuated BLM-evoked pulmonary lipid peroxidation,ferroptosis-characteristic mitochondrial ultrastructure and pulmonary epithelial death.The in vitro experiments showed that MMPs were decreased and mtROS were increased in BLM-exposed BEAS-2B cells.Mitoquinone(MitoQ),a mitochondria-targeted antioxidant,prevented BLM-induced MMP reduction and mitochondrial ROS elevation in BEAS-2B cells.The in vivo experiment found that MitoQ attenuated BLM-evoked GSH depletion and lipid peroxidation in mouse lungs.Moreover,MitoQ prevented BLM-induced ferroptosischaracteristic mitochondrial changes,pulmonary epithelial death and ALI.Conclusion:mitochondrial ROS are an initiator of BLM-induced pulmonary epithelial ferroptosis.Mitochondria-targeted antioxidants may be used as potential therapeutic agents for BLM-induced ALI.