Role And Mechanism Of Ferroptosis In Lung Injury Induced By Nanoplastics

Background and objectivesAs a global new pollutant,microplastics widely exist in environment and can decompose into nanoscale particles,accumulating in the environment.Micro（nano）plastics have posed great health threats to humans,which become a global public health problem that needs to be addressed.Polystyrene microplastics are one of the most common and representative microplastics in the environment.Respiratory inhalation is one of the main ways for the human to be exposed to atmospheric pollution,and the lungs are both exposed and target organs of micro（nano）plastics.In recent years,many scholars have pointed out that microplastics can damage the respiratory system and may induce chronic obstructive pulmonary disease（COPD）.Therefore,it is urgent to deeply understand the pulmonary toxicity effects and mechanisms of microplastics.As an important research breakthrough in cell death,ferroptosis has gradually become a research focus point in the studies on lung disease,which may be the key mechanism in lung injury caused by nanoplastics.In addition,organ-on-a-chip,as one of the top ten emerging technologies,has become the most cutting-edge revolutionary technology in biomedical research.Lung-on-a-chip can simulate the physiological activity and key structural characteristics of human lungs,and reflect the functional characteristics of lungs,which can be used to evaluate the harm of environmental pollutants and understand the pathogenesis.Therefore,the present study focused on the pulmonary toxicity of nanoplastics and selected polystyrene nanoplastics（PS-NPs）as the research object.Based on the mice respiratory exposure model and 2D cell mechanism research,this study innovatively combined the latest medical and industrial integration technology（lung-on-a-chip）to elucidate the nanoplastic induced lung injury and the potential mechanisms at different dimensions from genes,subcellular,cellular and tissues.The study provided theoretical basis and scientific clues for the prevention and treatment of lung injury caused by nanoplastics,and provided new ideas and effective technical means for the toxicological research of new environmental pollutants.Methods（1）The pulmonary toxicity in mice following inhalation exposure to nanoplastics The animal oronasal respiratory exposure tower was applied to establish the mice PS-NPs inhalation model.Three dose levels（5,12.5,31.25 mg/kg.bw/w）with theoretical and practical significance were set up.After the exposure,IVIS Spectrum system was applied to explore the distribution and accumulation of PS-NPs in mice.The effects of PS-NPs on mice pulmonary function were monitored by a whole-body plethysmography system.The general health of the mice was assessed by measurements of body weight and lung coefficients.In addition,serum,alveolar lavage fluid（BALF）and lung tissues of mice were obtained for subsequent studies.The pulmonary and systemic toxicity caused by PS-NPs in mice were assessed by examining the levels of oxidative stress,inflammatory responses,and cytotoxic effects.Histopathology and markers responsible for lung injury were observed to evaluate the lung injury.The effects of PS-NPs on subcellular structures were observed by transmission electron microscopy（TEM）and by detecting the mitochondrial fission fusion associated proteins DRP1,MFN1,MFN2,and VDAC1 as well as the endoplasmic reticulum stress markers BIP and CHOP.Meanwhile,correlation chord diagrams were applied to understand the association between lung injury and local/systemic toxicity.The transcriptome sequencing was performed to explore the potential mechanism of lung injury induced by PS-NPs.Finally,an adverse outcome pathway（AOP）framework for lung injury by PS-NPs was constructed based on the experimental results of this study and published experimental evidence.In summary,we established acute,subacute,and subchronic PS-NPs inhalation models in mice to preliminarily reveal the lung injury caused by nanoplastics and the underlying mechanisms from the whole animal level.（2）Involvement of ferroptosis in nanoplastics induced lung injuryThis study focused on ferroptosis,applying two lung epithelial cells as well as the mice Fer-1 treatment model to elucidate the role of ferroptosis in nanoplastic induced lung injury.CCK8 detected cell viability and established a dose-response relationship to obtain the experimental dose in vitro.The distribution and accumulation of nanoplastics in cells were evaluated by the flow cytometry and fluorescence microscopy.The oxidative and anti-oxidative balance status was evaluated by detecting ROS,superoxide anion,and oxidoreductase.We further evaluated protease and anti-protease balance by transcriptome sequencing and AAT expression.Then,the cell viability,iron content,lipid peroxidation level,and expression of ferroptosis key genes were examined in the presence or absence of the ferroptosis inhibitors DFO or Fer-1.Meanwhile,we compared with the protective effects of necroptosis inhibitor Nec-1 or apoptosis inhibitor z VAD to elucidate the ferroptosis was the main cell death induced by PS-NPs.Furthermore,antioxidant NAC was applied to explore the effects of oxidative stress on ferroptosis induced by PS-NPs.Finally,blank control group,Fer-1 control group,PS-NPs high-dose group,and Fer-1 treatment group were set up to perform a 4-week subacute inhalation exposure in mice.The pulmonary function,oxidative stress,inflammatory response,lung injury markers,and ferroptosis were measured to further elucidate the role of ferroptosis in PS-NPs induced lung injury from the animal level.（3）Ferroptosis induced by nanoplastics mediated by oxidative stress/subcellular-regulated ferritinophagyThe effects of PS-NPs on mitochondrial structure and morphology were evaluated by transmission electron microscope（TEM）.Mitochondrial dysfunction was detected by measuring mitochondrial oxygen consumption rate（OCR）,m RNA levels of mitochondrial respiratory chain complexes I,III,IV and V components,and mitochondrial NAD⁺transporter SLC25A51 expression,as well as mitochondrial reactive oxygen species（mt ROS）content,membrane potential changes（MMP）,intramitochondrial iron content,and lipid oxidation levels.The mitochondrial fission fusion proteins DRP1,MFN1,MFN2,OPA1,and FIS1 were tested to explore possible mitochondrial dynamics disorders.Besides,calcium homeostasis and endoplasmic reticulum markers BIP,CHOP and Caspase-12 were detected to explore the possible endoplasmic reticulum（ER）stress induced by nanoplastics.In the presence or absence of NAC,the mitochondria targeted antioxidant Mito-TEMPO,or the ER stress inhibitor4-BPA,the subcellular function and ferroptosis were detected to explore the role of the oxidative stress-subcellular axis in PS-NPs induced ferroptosis.We further examine the effects of PS-NPs on autophagosomes by electron microscopy.The change tendency of LC3II/I and p62 were observed at different doses of PS-NPs with different exposure duration,combined with the effects of autophagy inhibitors 3-MA,Baf A1 and PE,to clarify the activation of autophagy by PS-NPs.In the presence or absence of autophagy inhibitors,the cell viability,labile iron pool,and lipid peroxidation levels were detected to elucidate that nanoplastics could induce autophagy-dependent ferroptosis.The correlation of ferritin and NCOA4 was analyzed by protein expression detection.Meanwhile,immunofluorescence confirmed that ferritin and NCOA4 co-localized with LC3.In the presence or absence of autophagy inhibitors,NCOA4 and ferritin expression were detected to clarify that nanoplastics could disrupt iron homeostasis via autophagy.Lastly,the si-NCOA4 cell lines were constructed to measure cell viability,free iron concentration,ROS production,ferritin expression,lipid peroxidation and AAT levels,which revealed that nanoplastics induced ferroptosis through NCOA4-mediated ferrinophagy.（4）Role and mechanism of ferroptosis in lung injury induced by nanoplastics based on the lung-on-a-chipAccording to the time axis of organ-on-a-chip construction,human lung epithelial cells HPAEpi C,vascular endothelial cells HUVEC and macrophage were sequentially seeded into the lung-on-chips and connected with an automated culture system for dynamic culture at an air liquid interface.The lung-on-chips were characterized by cell live/dead staining,three-dimensional imaging,tight junction proteins,and transmembrane electrical resistance（TEER）values.To establish an environmental toxicology platform,various pollutants were selected to confirm the stability and fidelity of the lung-on-a-chip,and determine the indicators of lung injury at tissue level.Cell viability,TEER values,permeability,inflammation,oxidative stress and AAT function were measure to evaluate lung injury.Then,different concentrations of PS-NPs were treated lung-on-a-chip to detect cell viability,TEER values,permeability,inflammatory factors,ROS production,monocyte adhesion,and AAT expression.Besides,PS-NPs uptake by different cells in the lung-on-a-chip was detected by flow cytometry to observe the transport processes of PS-NPs.In addition,iron ion content and GPX4 expression changes of the lung-on-a-chip were detected.After ferroptosis inhibitors Fer-1 pretreatment,the cellular activity,barrier function,inflammatory response,oxidative stress and AAT expression were examined to explore the role of ferroptosis in PS-NPs induced lung injury at the tissue level.Finally,the results from the mice models,cellular mechanism study,and results of lung-on-a-chip were integrated to refine and evaluate the AOP framework.Results（1）The pulmonary toxicity in mice following inhalation exposure to nanoplastics After exposure,PS-NPs were distributed and accumulated in the mice lungs.After acute,subacute,and subchronic exposure,PS-NPs induced excessive ROS production locally and systemically in a dose-dependent manner,and promoted oxidative stress continuously at different exposure duration.The levels of cytokines（IL-6,MCP-1 and TNF-α）and cytotoxic indicators were significantly increased with a good dose-and time-response relationship.With increasing exposure time and concentration of PS-NPs,the lung function of mice decreased with increased respiratory rates,decreased EF50values and increased Penh values.The airway and alveolar morphology and structure of lung tissues changed obviously,accompanied by inflammatory cell infiltration.Lung injury markers MMP-9 expression was significantly upregulated while AAT expression was significantly downregulated,resulting in a protease-antiprotease imbalance.Above results showed that PS-NPs could induce COPD-like lung injury.In addition,high-dose PS-NPs exposure caused swelling of the endoplasmic reticulum and mitochondria,and blurring or disappearance of mitochondrial ridges.PS-NPs also caused obvious changes in the mitochondrial-related proteins DRP1,MFN2,VDAC1 as well as ER stress-related proteins BIP and CHOP,suggesting that PS-NPs promoted mitochondrial damage and triggered ER stress.Transcriptome sequencing of lung tissue identified that differential genes significantly enriched on COPD,and ferroptosis was the potential important mechanism.In addition,after acute,subacute,and subchronic exposures,MDA and iron levels were significantly increased in the lung tissues with a dose-dependent manner.The expression of GPX4,NCOA4 and ferritin expression were also decreased.Finally,an AOP framework for lung injury induced by PS-NPs was construct,revealing that ROS generation by PS-NPs was the molecular initiating events,oxidative stress,inflammatory response,and subcellular dysfunction were the key events,and lung dysfunction was the adverse outcome.（2）Involvement of ferroptosis in nanoplastics induced lung injuryIn vitro 2D cell experiments revealed that PS-NPs could significantly reduce cell viability in both lung epithelia cells with dose-and time-response relationships.We chose 7.5,15,and 30μg/cm² for subsequent experiments.PS-NPs could be internalized by lung epithelial cells,accumulating in the cytoplasm in a time-and dose-dependent manner.PS-NPs could induce ROS produ ction that disrupted the lung cell redox balance,leading to oxidative stress.Transcriptome sequencing identified that differentially expressed genes were enriched on COPD and ferroptosis pathways.Combined with the comparison with the protective effects of Nec-1 and z VAD,it was suggested that ferroptosis was one of the main ways of cell death induced by PS-NPs.Besides,PS-NPs could significantly increase intracellular Fe²⁺content and variable iron pool levels,cause glutathione depletion,increase lipid peroxidation and alter the expression of ferroptosis genes.However,after pretreatment with ferroptosis inhibitors Fer-1 and DFO,characteristic changes of ferroptosis were lighten,including iron overload,glutathione depletion and lipid peroxidation as well ferroptosis genes and AAT alterations,indicating that ferroptosis involved in the lung injury induced by PS-NPs.In addition,we further verified that oxidative stress induced by PS-NPs promoted the ferroptosis by pretreatment with antioxidant NAC.Finally,in the mice subacute model,we scavenged lipid peroxidation to inhibit ferroptosis by intraperitoneal injection of Fer-1.The results showed that Fer-1 could attenuate ferroptosis caused by PS-NPs,and improve pulmonary function,alleviate PS-NPs induced oxidative stress,inflammatory response,cytotoxicity as well as lung tissue damage.（3）Ferroptosis induced by nanoplastics mediated by oxidative stress/subcellular-regulated ferritinophagyTEM images were observed that PS-NPs could damage mitochondrial structure.After exposure to PS-NPs for 24h,mitochondrial function was impaired,showing that OCR,mitochondrial respiratory chain complexes as well as the NAD⁺transporter SLC25A51were suppressed with increasing exposure concentrations.PS-NPs also caused mitochondrial mt ROS elevation,loss of membrane potential,mitochondrial iron overload and lipid peroxidation.Besides,imbalances in mitochondrial dynamics were induced by PS-NPs,including elevated expression of fission protein ser616,FIS1,and downregulation of fusion proteins MFN1 and MFN2 as well as outer membrane proteins VDAC1 and TOMM20,which predisposed to mitochondrial fission.Pretreatment with NAC and Mito-TEMPO could significantly alleviate mitochondrial disorders and ferroptosis caused by PS-NPs,indicating that PS-NPs induced ferroptosis by damaging mitochondria through oxidative stress.In addition,PS-NPs exposure could lead to intracellular calcium imbalance and aggregation of unfolded proteins,which triggered ER stress.The ER stress induced by PS-NPs could be partially alleviated and the ferroptosis was also significantly reduced by pretreatment with NAC and 4-BPA,indicating that the oxidative stress-mediated ER stress was involved in the ferroptosis induced by PS-NPs.During PS-NPs exposure,autophagy was activated.Autophagy was confirmed to promote PS-NPs induced ferroptosis by autophagy inhibitors co-treatment.We found that ferritin and LC3 were co-localized and PS-NPs induced ferritin autophagy-dependent degradation.Meanwhile,pretreatment with NAC,Mito-TEMPO and 4-PBA could decrease the intracellular autophagosome accumulation and inhibit autophagy to restore NCOA4 and ferritin expression,indicating that PS-NPs induce autophagy and degrade ferritin via the oxidative stress-subcellular signaling axis.In addition,knockdown of NCOA4 could block ferritin degradation caused by PS-NPs,and improve cell viability,inhibit ROS production,alleviate ferroptosis and lipid peroxidation,and restore AAT expression,indicating that PS-NPs could induce NCOA4-mediated ferritinophagy,which in turn increased intracellular iron ions to cause ferroptosis and promote the lung injury.（4）Role and mechanism of ferroptosis in lung injury induced by nanoplastics based on the lung-on-a-chipThe lung-on-a-chip was constructed following the timeline to simulate the alveolar–capillary barrier structure in vivo.Under ALI culture,the cells grew well and gradually formed confluent cell layers,and the TEER values were maintained at 40Ω*cm²,which could be stably cultured for more than two weeks.Modeling human lung-specific responses were performed using a panel of representative environmental pollutants,including bioaerosols,chemical pollutants and nanoparticles,to evaluate the reliability,practicability,and stability of the toxicological application on chips.Moreover,PS-NPs could cause barrier dysfunction,burst inflammatory response,oxidative stress,AAT function impaired,and accumulated in lung cells and enter blood,which might increase the risk for COPD.Moreover,PS-NPs resulted in elevated iron ion content and downregulated GPX4 expression in lung-on-chips.Fortunately,pretreatment with Fer-1 could reduce ferroptosis,alleviated inflammatory response and oxidative stress,restored barrier function and AAT expression,clarifying the role of ferroptosis in lung injury induced by nanoplastics.Finally,we refined the AOP framework through direct evidence among key events to delineate the network of adverse outcome pathways of PS-NPs induced lung injury.The AOP emphasized that starting with the molecular initiating events of excessive ROS,followed by the key toxic pathway of oxidative stress-ferroptosis,lead to adverse outcome of lung injury,which had a high confidence.ConclusionsIn this study,we focused on a novel perspective on ferroptosis to deeply elucidate the effects and mechanisms of nanoplastic induced lung injury in the mice inhalation models,2D cell models and 3D organ-on-a-chip levels.During nanoplastics exposure,nanoplastics disrupted subcellular structure and function through oxidative stress,then induced ferritinophagy to cause iron imbalance,and further promoted lipid peroxidation,which led to ferroptosis,promoted lung injury,and increased the risk of COPD.This study provided scientific clues for the therapeutic targets and prevention and treatment measures of lung injury induced by nanoplastics,and offered new ideas and effective technical means for the toxicological evaluation of environmental pollutants.