[Study on cellular repressor of E1A-stimulated genes regulating autophagy and polarization of alveolar macrophages in sepsis-induced acute respiratory distress syndrome and its clinical value].
To investigate the regulatory effects of cellular repressor of E1A-stimulated gene (CREG) on autophagy and polarization of alveolar macrophages in sepsis-induced acute respiratory distress syndrome (ARDS) and its clinical value.
1) Cell experiment: Mouse monocyte/macrophage cell line RAW264.7 was cultured in vitro. Cells in logarithmic growth phase were induced to differentiate into alveolar macrophages. The alveolar macrophages were divided into four groups: the blank control group was cultured with complete medium only; the lipopolysaccharide (LPS) group was stimulated with 5 mg/L LPS for 24 hours to establish the sepsis-induced ARDS model; the CREG overexpression group was transfected with 2 mg CREG overexpression plasmid pLNCX2-CREG for 6 hours, followed by 5 mg/L LPS stimulation for 24 hours; the CREG interference group was transfected with 2 μg CREG interference plasmid pSM2-siCREG for 6 hours, followed by 5 mg/L LPS stimulation for 24 hours. Western blotting was used to detect the expression of autophagy markers [autophagy initiation key protein Beclin1, microtubule-associated protein 1 light chain 3 (LC3), and autophagic substrate protein p62]. Flow cytometry was used to detect the expression of macrophage polarization markers [M1-type characteristic marker cluster of differentiation 86 (CD86) and M2-type characteristic marker CD206]. 2) Clinical trial: A prospective case-control study was conducted in patients with sepsis admitted to the respiratory intensive care unit of The First Affiliated Hospital of Hebei North University from January to December 2024. Patients were divided into sepsis with ARDS group and sepsis without ARDS group based on whether they developed ARDS within 72 hours after enrollment. In addition, healthy volunteers who underwent health check-ups at the hospital during the same period were selected as the controls. Demographic data including gender, age, Acute Physiology And Chronic Health Evaluation II (APACHE II) score, oxygenation index (PaO2/FiO2), and laboratory parameters were collected for each group, as well as the sites of infection for the septic patients. Serum levels of CREG and inflammatory markers [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), procalcitonin (PCT), and C-reactive protein (CRP)] were measured using enzyme-linked immunosorbent assay (ELISA). The differences in the above indicators were compared among groups. Multivariate Logistic regression analysis was used to identify independent influencing factors for sepsis complicated with ARDS. Receiver operator characteristic curve (ROC curve) was plotted to evaluate the predictive value of CREG for sepsis complicated with ARDS.
1) Cell experiment results: Compared with the blank control group, the expressions of Beclin1, LC3-II/LC3-I ratio, and CD206 were decreased in each LPS group [Beclin1 protein (Beclin1/β-actin): 0.32±0.05 vs. 0.87±0.09, LC3-II/LC3-I ratio: 0.41±0.06 vs. 1.24±0.11, CD206: (27.14±3.52)% vs. (51.78±5.91)%, all P<0.05], while p62 and CD86 expressions were increased [p62 protein (p62/β-actin): 1.58±0.13 vs. 0.53±0.07, CD86: (38.35±4.67)% vs. (18.26±3.24)%, both P<0.05]. CREG overexpression significantly improved the above autophagy and polarization indicators, whereas CREG interference further exacerbated these indicators (all P<0.05). 2) Clinical trial results: Ultimately, 40 patients were included in the sepsis with ARDS group, 60 in the sepsis without ARDS group, and 20 in the healthy control group. There were no statistically significant differences in gender, age among the three groups, nor in the distribution of infection sites between the sepsis with ARDS and sepsis without ARDS groups (all P>0.05). Compared with the healthy control group, the patients with sepsis showed aggravated systemic inflammatory burden. Compared with the sepsis without ARDS group, the sepsis with ARDS group exhibited more severe disease, with lower PaO2/FiO2 and CREG levels, and higher levels of inflammatory markers (all P<0.05). As APACHE II score increased, serum CREG levels decreased progressively while inflammatory marker levels increased progressively. Multivariate Logistic regression analysis showed that elevated APACHE II score and inflammatory markers were independent risk factors for sepsis complicated with ARDS [all odds ratio (OR) >1, all P<0.05], while elevated CREG was a protective factor [OR=0.385, 95% confidence interval (95%CI) was 0.344-0.432, P=0.015]. ROC curve analysis showed that the area under the ROC curve (AUC) of CREG for predicting sepsis complicated with ARDS was 0.806 (95%CI was 0.713-0.899); at the optimal cut-off value of 7.85 μg/L, the sensitivity was 71.43% and the specificity was 78.57%.
CREG exerts a protective role in sepsis-induced ARDS by positively regulating alveolar macrophage autophagy activity and correcting M1/M2 polarization imbalance. Elevated serum CREG is a protective factor for sepsis complicated with ARDS and has early predictive value for sepsis-induced ARDS.
1) Cell experiment: Mouse monocyte/macrophage cell line RAW264.7 was cultured in vitro. Cells in logarithmic growth phase were induced to differentiate into alveolar macrophages. The alveolar macrophages were divided into four groups: the blank control group was cultured with complete medium only; the lipopolysaccharide (LPS) group was stimulated with 5 mg/L LPS for 24 hours to establish the sepsis-induced ARDS model; the CREG overexpression group was transfected with 2 mg CREG overexpression plasmid pLNCX2-CREG for 6 hours, followed by 5 mg/L LPS stimulation for 24 hours; the CREG interference group was transfected with 2 μg CREG interference plasmid pSM2-siCREG for 6 hours, followed by 5 mg/L LPS stimulation for 24 hours. Western blotting was used to detect the expression of autophagy markers [autophagy initiation key protein Beclin1, microtubule-associated protein 1 light chain 3 (LC3), and autophagic substrate protein p62]. Flow cytometry was used to detect the expression of macrophage polarization markers [M1-type characteristic marker cluster of differentiation 86 (CD86) and M2-type characteristic marker CD206]. 2) Clinical trial: A prospective case-control study was conducted in patients with sepsis admitted to the respiratory intensive care unit of The First Affiliated Hospital of Hebei North University from January to December 2024. Patients were divided into sepsis with ARDS group and sepsis without ARDS group based on whether they developed ARDS within 72 hours after enrollment. In addition, healthy volunteers who underwent health check-ups at the hospital during the same period were selected as the controls. Demographic data including gender, age, Acute Physiology And Chronic Health Evaluation II (APACHE II) score, oxygenation index (PaO2/FiO2), and laboratory parameters were collected for each group, as well as the sites of infection for the septic patients. Serum levels of CREG and inflammatory markers [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), procalcitonin (PCT), and C-reactive protein (CRP)] were measured using enzyme-linked immunosorbent assay (ELISA). The differences in the above indicators were compared among groups. Multivariate Logistic regression analysis was used to identify independent influencing factors for sepsis complicated with ARDS. Receiver operator characteristic curve (ROC curve) was plotted to evaluate the predictive value of CREG for sepsis complicated with ARDS.
1) Cell experiment results: Compared with the blank control group, the expressions of Beclin1, LC3-II/LC3-I ratio, and CD206 were decreased in each LPS group [Beclin1 protein (Beclin1/β-actin): 0.32±0.05 vs. 0.87±0.09, LC3-II/LC3-I ratio: 0.41±0.06 vs. 1.24±0.11, CD206: (27.14±3.52)% vs. (51.78±5.91)%, all P<0.05], while p62 and CD86 expressions were increased [p62 protein (p62/β-actin): 1.58±0.13 vs. 0.53±0.07, CD86: (38.35±4.67)% vs. (18.26±3.24)%, both P<0.05]. CREG overexpression significantly improved the above autophagy and polarization indicators, whereas CREG interference further exacerbated these indicators (all P<0.05). 2) Clinical trial results: Ultimately, 40 patients were included in the sepsis with ARDS group, 60 in the sepsis without ARDS group, and 20 in the healthy control group. There were no statistically significant differences in gender, age among the three groups, nor in the distribution of infection sites between the sepsis with ARDS and sepsis without ARDS groups (all P>0.05). Compared with the healthy control group, the patients with sepsis showed aggravated systemic inflammatory burden. Compared with the sepsis without ARDS group, the sepsis with ARDS group exhibited more severe disease, with lower PaO2/FiO2 and CREG levels, and higher levels of inflammatory markers (all P<0.05). As APACHE II score increased, serum CREG levels decreased progressively while inflammatory marker levels increased progressively. Multivariate Logistic regression analysis showed that elevated APACHE II score and inflammatory markers were independent risk factors for sepsis complicated with ARDS [all odds ratio (OR) >1, all P<0.05], while elevated CREG was a protective factor [OR=0.385, 95% confidence interval (95%CI) was 0.344-0.432, P=0.015]. ROC curve analysis showed that the area under the ROC curve (AUC) of CREG for predicting sepsis complicated with ARDS was 0.806 (95%CI was 0.713-0.899); at the optimal cut-off value of 7.85 μg/L, the sensitivity was 71.43% and the specificity was 78.57%.
CREG exerts a protective role in sepsis-induced ARDS by positively regulating alveolar macrophage autophagy activity and correcting M1/M2 polarization imbalance. Elevated serum CREG is a protective factor for sepsis complicated with ARDS and has early predictive value for sepsis-induced ARDS.