Mechanistic Investigation Of Autophagy Dysfunction In Alveolar Type Ⅱ Cells During Multi-walled Carbon Nanotube-induced Pulmonary Fibrosis

Background and objectiveThe production and use of nanomaterials have led to a pressing need to address the impact of nanomaterial pollution on human health and the environment.Multi-walled carbon nanotubes（MWCNTs）,with their excellent mechanical,electrical,and magnetic properties,are widely used in fields such as electronics,structural engineering,medicine,and hydrogen fuel cells.However,as the scope and production of MWCNTs continue to expand,the health risks posed by exposure to MWCNTs through environmental and occupational pathways have become a significant public health concern that cannot be ignored.Inhalation exposure to carbon nanotubes can cause pulmonary inflammation and fibrosis,but the underlying mechanisms are not yet fully understood.Therefore,this study focused on exploring the target and toxic mechanism of pulmonary fibrosis induced by inhalation exposure to MWCNTs,in order to provide scientific and reasonable occupational protection recommendations for occupational exposure to MWCNTs.MethodsA pulmonary fibrosis model was established by a single intratracheal instillation of multi-walled carbon nanotubes（MWCNTs）at a dose of 2 mg/kg in C57BL/6J mice.After120 days of exposure,lung pathological changes were observed by HE staining and Masson’s trichrome staining.The expression levels of inflammatory cytokines and pulmonary fibrosis markers in bronchoalveolar lavage fluid（BALF）and serum were detected by Enzyme-Linked Immunosorbent Assay（ELISA）.The changes of lung function induced by MWCNTs were detected by pulmonary function analyzer.The level of dipalmitoyl phosphatidylcholine（DPPC）in BALF was quantitatively analyzed using Ultra High-Performance Liquid Chromatography-Orbitrap Mass Spectrometry（UHPLC-Orbitrap MS）.The effects of MWCNT inhalation on the fibrosis and autophagy-related pathways in mouse lung tissue were analyzed using transcriptome sequencing.Furthermore,changes in the expression of autophagy-related proteins,LC3B and P62,in mouse lung tissue were further detected by immunofluorescence and Western Blot.Primary alveolar type II（AT2）cells from mice were exposed to MWCNTs at concentrations of 10μg/m L and 25μg/m L.The ultrastructure changes of primary AT2 cells after MWCNT exposure were observed by transmission electron microscopy.The expression levels of autophagy-related genes and proteins in AT2 cells were detected by Western Blot and RT-PCR.The autophagy flux and lysosomal p H changes in primary AT2cells after MWCNT treatment were detected using a dual-labeled adenovirus（m RFP-GFP-LC3）and a lysosomal probe（Lysosensor）.Results1.Exposure to MWCNTs caused pulmonary and systemic inflammation,as well as lung fibrosis,in mice.The levels of inflammatory cytokines such as IL-6,TNF-α,and IL-1β were significantly increased in the BALF and serum.Lung tissue pathology showed accumulation of MWCNTs and fibrotic nodules,with an increase in lung injury pathology score.2.Mice exposed to MWCNTs exhibited restrictive ventilatory dysfunction and increased lung surface tension.Lung function results showed a decrease in lung volume,as well as Forced Vital Capacity（FVC）and Forced Expiratory Volume 0.1s(FEV_0.1).3.Exposure to MWCNTs caused lamellar body（LB）damage and autophagy inhibition in AT2 cells in mice.Electron microscopy showed vacuolization of the LB responsible for secreting pulmonary surfactant.The content of the main components of pulmonary surfactant,DPPC and Pulmonary Surfactant Protein-C（SP-C）,was reduced in the BALF. Transcriptome sequencing of mouse lung tissue suggested disturbance of fibrosis and autophagy-related pathways,with upregulation of autophagy-related proteins LC3B and P62.4.In primary AT2 cells,exposure to MWCNTs resulted in vacuolization of the lamellar body and upregulation of autophagy-related proteins.dual-labeled adenovirus（m RFP-GFP-LC3）showed that autophagy flow was blocked,and fusion between autophagosomes and lysosomes was inhibited.5.Lysosensor results showed that exposure to MWCNTs led to lysosomal acidification,with downregulation of the gene expression of lysosomal-associated proteases Ctsa and Ctsd,leading to imbalance in lysosomal homeostasis.ConclusionIn vivo studies showed that MWCNTs increased lung surface tension by affecting surfactant secretion in mice after inhalation exposure.Further studies showed that MWCNTs induce lamellar body dysfunction in primary AT2 cells responsible for the secretion of lung surfactants by inhibiting the fusion of autophagosome and lysosome,blocking autophagy flux.The findings of this study provide a potential therapeutic target for MWCNT-induced pulmonary fibrosis,as well as novel insights into the pathogenesis of occupational injury caused by exposure to MWCNTs.