Ferroptosis of bronchial epithelial cells induced by pseudomonas aeruginosa infection and the immunomodulatory mechanism of sodium butyrate
ZHOU Luan1, HE Yifan1, 2, ZHAO Yimin3, REN Xikai1,GENG Xuan1, HUANG Nan1, SU Miaoshang1.
1.Department of Pediatric Respiratory Diseases, the Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, Wenzhou 325027, China;2.Department of Endocrinology, the First People’s Hospital of Longwan District, Wenzhou 325024, China; 3.The Second Medical College, Wenzhou Medical University, Wenzhou 325035, China
ZHOU Luan,HE Yifan,ZHAO Yimin, et al. Ferroptosis of bronchial epithelial cells induced by pseudomonas aeruginosa infection and the immunomodulatory mechanism of sodium butyrate[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2024, 54(7): 554-562.
Abstract：Objective: To investigate the inductive effect of pseudomonas aeruginosa (PA) infection on ferroptosis and inflammation in human bronchial epithelial cells (BEAS-2B) and the immunomodulatory mechanism of sodium butyrate (NaB). Methods: In the in vitro BEAS-2B cell culture system, different concentrations of NaB were added to screen the safe concentration and the suitable time by CCK8 assay. Changes in the expression of acyl-CoA synthetase long-chain family member 4 (ACSL4) and phosphorylated protein kinase B (P-AKT) were detected by Western blot to determine the concentration of NaB. BEAS-2B cells infected with PA (MOI=1), and after different period of time, cell viability was measured using CCK-8 assay; changes in cell morphology were observed by inverted microscopy and mitochondrial morphology were observed by transmission electron microscopy, and intracellular Fe2+ level was detected by Fe2+ fluorescence probe. After the pretreatment with NaB, the intracellular lipid peroxidation level was detected by BODIPY 581/591 C11 fluorescence probe and changes in the expression of ACSL4, P-AKT and glutathione peroxidase 4 (GPX4) were detected by Western blot.After the pretreatment with pioglitazone (PIO), the inhibitory effect on ACSL4 was verified by Western blot, and the inflammatory factors in different groups were detected by ELISA. Results: The safe concentration of NaB for BEAS-2B cells was below 2.5 mmol/L, and with the concentration of 2.5 mmol/L treated for 36 hours, the protein levels of ACSL4 and P-AKT protein were decreased significantly (P<0.05). After PA infection, cell viability was reduced in BEAS-2B cells (P<0.05). The cell became round in shape, mitochondria were the smaller and mitochondrial crista decreased or even disappeared in the ferroptotic cells; the intracellular Fe2+ and lipid peroxidation levels were significantly increased (P<0.05) and the protein levels of ACSL4 and P-AKT were increased (P<0.05), while the protein level of GPX4 was significantly decreased (P<0.05). Compared with the PA group, the protein levels of ACSL4 and P-AKT were decreased with the pretreatment of NaB (P<0.05),which, however, promoted the decreased levels of GPX4 and the accumulation of lipid peroxides. Compared with the control group, the ELISA results showed that the levels of IL-6, IL-8, IL-1β and TNF-α were increased significantly after PA infection (P<0.05), and the level of IL-10 was slightly decreased (P<0.05). After the pretreatment with NaB or PIO, the level of proinflammatory factors was significantly decreased (P<0.05), while the level of IL-10 showed no statistical significance (P>0.05). Conclusion: PA infection leads to the accumulation of Fe2+ and lipid peroxides in bronchial epithelial cells, which induces ferroptosis and inflammation, and NaB may play an immunomodulatory role by inhibiting the pathways of ACSL4 and AKT.
