Fig 1: OLFM4 inhibited the pro-inflammatory responses of lung epithelial cells by blocking NF-κB activation. (A) MLE-12 cells were transfected with OLFM4 plasmids or negative control (NC) for 24 h before LPS stimulation. Expression of IL-6, CCL2, CXCL1, and LCN2 in the supernatants of MLE-12 cells was measured using ELISA. *p < 0.05 versus NC group, **p < 0.01 versus NC group, #p < 0.05 versus NC+LPS group, ##p < 0.01 versus NC+LPS group. (B) OLFM4 expression and the level of phosphorylated NF-κB/P65 in MLE-12 cells were examined by Western blot. (C) BEAS-2B cells were pretreated with human recombinant OLFM4 (500 ng/mL or 1 µg/mL) for 30 min before stimulated with LPS. The mRNA expression of IL-6, CXCL1, IL-8, and LCN2 was measured by real-time RT-qPCR. *p < 0.05 versus CT group, **p < 0.01 versus CT group, #p < 0.05 versus LPS group, ##p < 0.01 versus LPS group. (D) OLFM4 expression and the level of phosphorylated NF-κB/P65 in BEAS-2B cells were examined by Western blot.
Fig 2: LDHA inhibition effectively alleviated LPS-induced inflammation in lung epithelial cells. (A) MLE-12 cells were pre-treated with FX-11 (LDHA inhibitor) for 30 min before LPS stimulation. The concentrations of IL-6, CCL2, CXCL1, and LCN2 in supernatants were measured using ELISA. N=3. (B) MLE-12 cells were pre-treated with FX-11 (LDHA inhibitor) for 30 min before LPS stimulation. The expression of phosphorylated NF-?B/P65, phosphorylated LDHA (p-LDHA) and LDHA in MLE-12 cells was detected by Western blot. (C) BEAS-2B cells were pre-treated with FX-11 for 30 min before stimulated with LPS. The mRNA levels of IL-6, CXCL1, IL-8, LCN2 were determined by real-time RT-qPCR. (D) BEAS-2B cells were pre-treated with FX-11 for 30 min before stimulated with LPS. The expression of phosphorylated NF-?B/P65, phosphorylated LDHA (p-LDHA) and LDHA in BEAS-2B cells was detected by Western blot. *p < 0.05 versus CT group, **p < 0.01 versus CT group, #p < 0.05 versus LPS group, ##p < 0.01 versus LPS group.
Fig 3: OLFM4 expression was different between septic and sepsis-induced ARDS patients. (A and B) Heatmap and statistical analyses of expression of the ten hub genes in the GSE66890 dataset. Red = upregulated. Blue = downregulated. **p < 0.01. (C) Analyses of ROC curves of critical DEGs in the GSE66890 dataset. ROC curves were generated and the area under the ROC was used to compare the ten genes in the sepsis group and sepsis-related ARDS group. Three DEGs showed AUC >0.7: 0.716 for OLFM4, 0.719 for LCN2, and 0.735 for BPI.
Fig 4: Validation of OLFM4 expression at the transcriptional and protein level. (A) Expression of ten critical genes was compared between healthy controls, septic patients, and sepsis-related ARDS patients by quantitative real-time PCR. Differences between two groups were analyzed. *p < 0.05, **p < 0.01 versus CT group, #p < 0.05 versus sepsis group. (B, C and D) Plasma OLFM4, LCN2 and BPI expressions were compared between healthy controls, septic patients, and sepsis-related ARDS patients using ELISA kit. **p < 0.01 versus control group, ***p < 0.001 versus control group, #p < 0.05 versus sepsis group. (E) Analyses of ROC curves of plasma OLFM4, LCN2 and BPI expression. ROC curves were generated and the area under the ROC was used to compare plasma OLFM4, LCN2 and BPI expressions in the sepsis group and sepsis-related ARDS group.
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