Mechanisms Of Lung Injury Induced By Acrolein In Vitro And In Vivo

Acrolein is a highly toxic α-β unsaturated aldehyde,which is widely found in the environment,food and water.It enters the human body mainly through skin contact,food intake and inhalation.Cigarette smoke is the main source of human exposure to acrolein,which is also one of the most dangerous toxic substances in cigarette smoke.Long-term smoking can induce a variety of respiratory diseases,and is the leading risk factor for chronic obstructive pulmonary disease(COPD).COPD is a common respiratory disease characterized by high morbidity,disability and mortality.Inhalation of acrolein is considered to be a key cause of respiratory diseases,and the lung is the main organ attacked by acrolein,but the mechanism of injury remains unclear.In view of the wide existence of acrolein,strong toxicity and high human exposure concentration,it is particularly important to explore the mechanism of its toxicity.The aims of this study were to investigate the mechanism of lung injury induced by acrolein in vitro and in vivo with cell biology,molecular biology,transcriptomics,bioinformatics and histopathology.At the cellular level,human bronchial epithelial cells(BEAS-2B)were studied to investigate the effects of cell damage and programmed death.(1)The cell viability and morphological changes after exposure to acrolein were detected,and the intracellular reactive oxygen species(ROS)level were measured.The key proteins expression of p38 MAPK and JNK pathways were detected,the nuclear translocation of NF-κB p65 and the release of inflammatory factors were measured,the oxidative stress injury and inflammatory response induced by acrolein were analyzed.The content of 8-hydroxydeoxyguanosine(8-OHd G),nuclear morphology,cell cycle distribution and the key proteins expression of ATM/Chk2 and ATR/Chk1 pathways were determined to analyze whether acrolein could activate the DNA damage response pathway.(2)Mitochondrial membrane potential(MMP),apoptosis rate,proteins expression of mitochondrial apoptosis pathway were detected,and the mechanism of cell apoptosis induced by acrolein was analyzed.Combining ferroptosis inhibitors and inducers,the content of iron ion,the activity of glutathione peroxidase 4(GPX4),the level of glutathione(GSH),the content of malondialdehyde(MDA),the release of lactate dehydrogenase(LDH)were investigated,and the protein expression of Nrf2/GSH/GPX4 pathway was detected to investigate whether acrolein could induce cell ferroptosis.At the transcriptional level,long non-coding RNA(lncRNA)was sequenced in the normal control group and the acrolein injured group to screen the target lncRNA and explore its regulatory effects.(1)The whole expression of lncRNA and mRNA in cells were analyzed,lncRNA and mRNA with significant differences were screened,and bioinformatics technology was used for overall analysis and functional prediction,so as to explore the possible pathways and biological processes involved in the toxic effects of acrolein.(2)The sequencing results were verified at the cell level,the target lncRNA was screened,and the lncRNA/mi RNA/mRNA regulatory axis was constructed.By knocking down the expression of target lncRNA,the changes in mi RNA and mRNA expression in the regulatory axis were detected,the interaction between target lncRNA and key proteins in the toxic mechanism of acrolein was predicted,and the changes in the expression of key proteins in the nuclear translocation of NF-κB p65 and Nrf2/GSH/GPX4 ferroptosis pathways were detected,the regulatory role of target lncRNA in the toxicity of acrolein were explored.At the animal level,the rat model of lung injury induced by aerosol inhalation of acrolein was established to explore the mechanism of lung injury induced by acrolein and the regulatory role of target lncRNA.(1)Lung morphological changes of rats were observed and lung index was calculated.Tissue section staining was used to detect lung structure,pulmonary fibrosis,iron content,and the genes expression related to lncRNA/mi RNA/mRNA regulatory axis were determined.The crucial protein expressions of p38 MAPK and JNK pathways and their downstream NF-κB p65 nuclear translocations were analyzed in lung tissue,the key protein expressions of DNA damage response,mitochondrial apoptosis and ferroptosis pathways were detected to explore the mechanism of lung injury induced by acrolein.(2)Target lncRNA were knocked down,the expression of target lncRNA in lung tissues and the expression changes of mi RNA and mRNA in the regulatory axis were detected.The differences of lung structure,pulmonary fibrosis and iron ion content in rats were compared,the crucial protein expression in the inflammation and ferroptosis pathways were detected in lung tissue,and the regulatory mechanism of lncRNA in lung injury induced by acrolein was explored.The results at the cellular level showed:(1)Acrolein(20,40 and 80 μM)induced the cell viability of BEAS-2B to 93.93 ± 4.80%,82.60 ± 2.95% and 58.07 ± 5.44% of the control group,respectively,and changed the cell morphology.Acrolein treatment increased intracellular ROS levels,activated p38 MAPK and JNK signaling pathways,promoted the nuclear translocation of NF-κB p65 and the release of inflammatory factors,and caused cellular oxidative stress and inflammation.Furthermore,acrolein caused oxidative damage to DNA,changed nuclear morphology,arrested G2/M cell cycle,and activated of ATM/Chk2 and ATR/Chk1 pathways,suggesting that acrolein could induce DNA damage and activate DNA damage response pathways.Molecular docking results showed that acrolein could spontaneously bind to DNA,indicating that there was an interaction between acrolein and DNA.(2)After exposure to acrolein,MMP decreased to 86.58 ± 2.54%,69.73 ± 2.61% and 55.38 ± 2.96% of the control group,respectively.The apoptosis rate was significantly increased(P < 0.01),and the Bax/Bcl-2 ratio,Cleared Caspase-9,and Cleared Caspase-3 were significantly increased(P < 0.01),indicating that acrolein activated the intrinsic mitochondrial apoptotic pathway.In addition,the intracellular iron content,MDA content and LDH release increased after acrolein exposure,while GSH content and GPX4 activity decreased.The expression of key proteins of Nrf2/GSH/GPX4 signaling transduction pathway was decreased,and the antioxidant capacity of cells was weakened,inducing ferroptosis.The results at the transcriptome sequencing level showed:(1)A total of 18,868 differenced lncRNAs and 15,639 differenced mRNAs were obtained after acrolein exposure.After screening,636 lncRNAs and 214 mRNAs were significantly different between the acrolein injured group and the control group,including 355 up-regulated lncRNAs,281 down-regulated lncRNAs,107 up-regulated mRNAs and 107down-regulated mRNAs.A comprehensive analysis of the Gene Ontology(GO)enrichment results of different lncRNAs and different mRNAs showed that acrolein treatment might affect tissue homeostasis,iron binding,DNA topological changes,MMP regulation and mitochondrial apoptosis pathway,etc.(2)LncRNA MIR155 HG was significantly highly expressed both at sequencing and cellular levels,and the predicted results indicated that it might interact with proteins related to inflammation,DNA damage response,mitochondrial apoptosis and ferroptosis pathways related to acrolein toxicity,and a regulatory loop of lncRNA MIR155HG/mi R-155-5p/CD200R1/NF-κB was constructed.Compared with acrolein injury group,the viability of BEAS-2B cells was significantly increased after lncRNA MIR155 HG knockdown(P < 0.01),the expression of mi R-155-5p was decreased,the expression of CD200R1 was increased,the ROS level was decreased,nuclear protein transposition of NF-κB p65 and the expression of inflammatory factors were decreased.The proteins expression of Nrf2/GSH/GPX4 pathway were increased,indicating that lncRNA MIR155 HG knockdown could relieve acrolein-induced cellular inflammation,improve cellular antioxidant capacity,and inhibit ferroptosis.The results at the animal level showed:(1)Acrolein induced lung injury in rats.The growth rate of body weight decreased,lung morphological abnormalities led to pulmonary edema,and acrolein also caused the destruction of lung tissue structure,the enlargement of alveolar space,and induced pulmonary fibrosis.Western blot results showed that acrolein exposure activated p38 MAPK and JNK pathways,induced nuclear translocation of NF-κB p65,promoted the expression of inflammatory factors,activated DNA damage response pathway and induced mitochondrial apoptosis.In addition,acrolein causes elevated levels of iron in lung tissue,triggering ferroptosis.(2)LncRNA MIR155 HG knockdown effectively improved lung injury induced by acrolein.After lncRNA MIR155 HG was knocked down,the lung tissue structure damage induced by acrolein was reduced,the alveolar space was reduced,the pulmonary edema and pulmonary fibrosis were improved,and the activation of NF-κB inflammatory pathway was inhibited.At the same time,the level of iron content in lung tissue was reduced,and ferroptosis was inhibited.In this study,based on the results of in vitro cell experiments and in vivo animal experiments combined with transcriptomic analysis,acrolein exposure could cause DNA damage in BEAS-2B cells and lung tissue,activate ATM/Chk2 and ATR/Chk1 DNA damage response and NF-κB inflammatory pathways,inhibit the Nrf2/GSH/GPX4 pathway of cellular antioxidant defense,trigger ferroptosis.LncRNA MIR155 HG might be a potential new target for the toxic inhibition of acrolein,which exerted toxic effects through lncRNA MIR155HG/mi R-155-5p/CD200R1/NF-κB regulatory loop.This study provides a new theory for the toxicity of acrolein-induced lung injury and its prevention and treatment,and provides experimental basis for the subsequent genomics and proteomics studies related to lung injury.At the same time,the potential molecular markers of toxic inhibition of acrolein were screened to provide new targets and strategies for the diagnosis and prevention of lung injury.