Annals of translational medicine · 2021
Vimentin regulation of autophagy activation in lung fibroblasts in response to lipopolysaccharide exposure in vitro.
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Pan Pan, Su Longxiang, Wang Xiaoting, Chai Wenzhao, Liu Dawei, Song Licheng, Xie Lixin
Аннотация
BACKGROUND: The activation and assembly of the NLRP3 inflammasome is dependent on the interaction between NLRP3 and the intermediate filament protein vimentin in an acute respiratory distress syndrome (ARDS) model. We investigated the role of vimentin in this process using human fetal lung (HFL-1) fibroblasts with vimentin transfer genes or gene knockdown and lipopolysaccharide (LPS) intervention.
METHODS: HFL-1 cells [con-vector + LPS, vimentin-pCMV3 (VIM-pCMV3), con-siRNA, and vimentin siRNA (VIM-siRNA)] were treated with LPS. An oxidative stress damage assessment, apoptosis analysis, and quantification of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 by enzyme linked immunosorbent assay (ELISA) were performed. Immunoblotting was used to reveal the autophagy pathway.
RESULTS: We demonstrated that in response to LPS vimentin expression was lower in the HFL-1 cells with the vimentin gene knocked down. Specifically, an increase in oxidative stress, a decrease in mitochondrial membrane potential, or an increase in calcium ion permeability resulted in an increase in the fibroblast apoptosis rate. In addition, the inflammatory response after vimentin gene knockout was upregulated, as indicated by higher levels of TNF-a, IL-1β, IL-6, and IL-10. Importantly, the mechanism of suppression of vimentin in the lung fibroblasts was caused by a decrease in autophagy, an increase in mitochondrial membrane protein, and a decrease in mitochondrial function, which may contribute to the augmented cellular injury generated during the response to LPS.
CONCLUSIONS: This study provides insights into whether vimentin may interfere with the inflammatory cascade by activating the autophagy pathway of mitochondrial lung fibroblasts in the early stage of acute lung injury (ALI).
METHODS: HFL-1 cells [con-vector + LPS, vimentin-pCMV3 (VIM-pCMV3), con-siRNA, and vimentin siRNA (VIM-siRNA)] were treated with LPS. An oxidative stress damage assessment, apoptosis analysis, and quantification of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-10 by enzyme linked immunosorbent assay (ELISA) were performed. Immunoblotting was used to reveal the autophagy pathway.
RESULTS: We demonstrated that in response to LPS vimentin expression was lower in the HFL-1 cells with the vimentin gene knocked down. Specifically, an increase in oxidative stress, a decrease in mitochondrial membrane potential, or an increase in calcium ion permeability resulted in an increase in the fibroblast apoptosis rate. In addition, the inflammatory response after vimentin gene knockout was upregulated, as indicated by higher levels of TNF-a, IL-1β, IL-6, and IL-10. Importantly, the mechanism of suppression of vimentin in the lung fibroblasts was caused by a decrease in autophagy, an increase in mitochondrial membrane protein, and a decrease in mitochondrial function, which may contribute to the augmented cellular injury generated during the response to LPS.
CONCLUSIONS: This study provides insights into whether vimentin may interfere with the inflammatory cascade by activating the autophagy pathway of mitochondrial lung fibroblasts in the early stage of acute lung injury (ALI).