Mechanism of ferroptosis induced by endoplasmic reticulum stress in sepsis related lung injury / 安徽医科大学学报

ABSTRACT

Objective @#To explore the mechanism of ferroptosis induced by endoplasmic reticulum stress (ERs ) in acute respiratory distress syndrome (ARDS) .@*Methods @#In order to determine the effects of LPS on oxidative stress and Fe2 + level of mouse capillary alveolar epithelial cells (MLE12 cells ) , the cells were treated with LPS (0 , 1 , 2 , 5 μg/ml) for 24 h . To verify the role of ferroptosis in lipopolysaccharide ( LPS)-induced cell death , MLE12 cells were divided into control ( Con ) group , iron removal inhibitor ( Fer-1) group , LPS group and LPS + Fer-1 group . LPS + Fer-1 group was pretreated with 10 μmol/L Fer-1 for 6 h , then the cells were exposed to 5 μg/ml LPS for 24 h . Con group was treated with solvent DMSO for 24 h . Fer-1 group was pretreated with 10 μmol/L Fer-1 for 6 h , and then treated with DMSO for 24 h . The cells in LPS group were exposed to 5 μg/ml LPS for 24 h . The MLE12 cells were divided into three groups : Con + Vector group , Con + sequence similarity family 134 mem ber B ( FAM134B ) group , LPS + Vector group and LPS + FAM134B group . After transfected with vector or FAM134B overexpression plasmid for 48 h , the cells were exposed or not exposed to 5 μg/ml LPS for 24 h . Cell viability was measured by CCK-8 . The levels of malondialdehyde (MDA) , glutathione and iron , the protein levels of ferroptosis markers [ cyclooxygenase 2(PTGS2) , glutathione peroxidase 4(GPX4)] and ERs markers [glucose reg ulatory protein 78( GRP78) , activated transcription factor 4 ( ATF4) and C/EBP homologous protein ( CHOP)] were measured in different groups . In order to further confirm the results of in vitro cell experiments , 40 mice were randomly divided into Con + Vector group , Con + FAM134B group , LPS + Vector group and LPS + FAM134B group , with 10 mice in each group . LPS induced sepsis models were established in LPS + Vector group and LPS + FAM134B group , and the levels of GPX4 and ERs in lung tissue were evaluated by immunofluorescence staining and protein blot. @*Results @#LPS treatment increased the levels of PTGS2 and MDA , and decreased the levels of GPX4 and GSH in MLE12 cells in a dose dependent manner. Compared with LPS group , the cell viability , GPX4 and GSH levels in LPS + Fer-1 group increased significantly (P < 0.05) , while the PTGS2 protein level and MDA level decreased significantly (P < 0.05) . Compared with LPS + Vector group , LPS + FAM134B group significantly increased cell viability (P < 0.05) , decreased PTGS2 protein level (P < 0.05) and increased GPX4 level ( P < 0.05) . At the same time , the level of MDA in LPS + FAM134B group was lower than that in LPS + Vector group (P < 0.05) , and the level of GSH was higher than that in LPS + Vector group (P < 0.05) . In animal experiment , compared with LPS + Vector group , the expression levels of 4 HNE , ATF4 and CHOP in lung tissue of LPS + FAM134B group decreased significantly ( P < 0.05 ) , and the expression levels of GPX4 , FAM134B group in creased significantly (P < 0.05) .@*Conclusion @#LPS induces ferroptosis and ERs in MLE12 cells in a dose depend ent manner. Activating the endoplasmic reticulum autophagy associated FAM134B receptor helps to inhibit ERs and alleviate cell ferroptosis .