Effects of estrogen-related receptor alpha on inflammatory response in pulmonary microvascular endothelial cells

Abstract

Objective To investigate the effect of estrogen-related receptor alpha(ERRα)on lipopolysaccharide-induced inflammatory response in rat pulmonary microvascular endothelial cells (PMVECs) and its mechanism. Methods PMVECs were cultured in vitro. When the cells were in the logarithmic growth phase, the cell were ransfected with lentivirus, and a stable low-expression ERRα cell line was constructed. The cells were divided into four groups: Ctr group (normal control group), Ctr+LPS group (normal cell+LPS treatment group), shERRα1 (shERRα1 gene knockdown group), and shERRα1+LPS group (shERRα1 gene knockdown +LPS treatment group). After 20 μg/mL LPS stimulated cells in the control group and shERRα1 group for 6, 12 and 24 h, cell counting kit-8 (cck-8) was used to detect the cell proliferation ability of each group, and enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of tumor necrosis factor alpha (TNF-α) and Interleukin-1β (IL-1β) in cell culture fluid. After 12 h LPS stimulation, the expression levels of ERRα and NF-κB related proteins (p-p65, p65, P-IKBα, IKBα) were measured by Western blot. Pairwise comparisons were performed with SNK-q test (two-tailed), and multiple-group comparisons were performed with one-way ANOVA. The non-parametric test of rank transformation was used when homogeneity of variance were not met. P value<0.05 was considered significantly different. Results Compared with the control group, ERRα expression in the shERRα group was significantly decreased (0.09±0.01 vs 0.15±0.01). At 6, 12 and 24 h after LPS stimulation, compared with the control group, the cell proliferation ability (%) of the shERRα1+LPS group was significantly reduced (99.68±4.53 vs 48.62±1.60) and the concentration of TNF-α (ng/mL) (15.76±3.38 vs 5 498.91±367.95) and IL-1β (ng/mL) (14.41±3.86 vs 6 014.92±277.33) in the cell culture supernatant were significantly increased. The change was most obvious after 12 h stimulation. Meanwhile the expression of p-p65 (0.30±0.50 vs 1.05±0.07) and p-IKBα (0.27±0.04 vs 0.77±0.06) were increased significantly, while the expression of IKBα (0.96±0.07 vs 0.14±0.04) was decreased significantly in the shERRα1+LPS group (all P<0.05). Conclusion ERRα gene attenuates LPS-induced inflammatory response in rat pulmonary microvascular endothelial cells by inhibiting NF-κB signaling pathway activation. Key words: Sepsis; Estrogen-related receptor alpha; NF-κB pathway; Gene knockdown