BRL-44408 maleate, the antagonist of α2A-adrenoceptor, attenuates endogenous lipopolysacchride-induced acute lung injury through inhibiting the mitogen-activated protein kinase kinase/extracellular regulated protein kinases signaling pathway in mice

Abstract

To explore the effects and mechanism of α2A-adrenergic receptor (α2A-AR) antagonist BRL-44408 maleate on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Sixty male C57BL/6 mice were randomly divided into three groups (n = 20): sham group, LPS group and BRL-44408 maleate pre-treated group (BRL+LPS group). The model of ALI was replicated by intratracheally administrated of LPS (5 mg/kg), and the mice in the sham group were received an equal volume of saline. Mice in the BRL+LPS group were treated with additionally BRL-44408 maleate (5 mg/kg, i.p) at 4 hours before LPS administration. The mice were sacrificed at 6 hours and 24 hours after LPS administration in each group. Among them, 5 mice were used to collect the bronchoalveolar lavage fluid (BALF) and the other 5 mice were sacrificed for lung tissues. The levels of norepinephrine (NE), tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-10) in BALF were measured by enzyme linked immunosorbent assay (ELISA). The level of protein in BALF was measured by bicinchoninic acid (BCA) method. The histopathological changes and wet/dry (W/D) ratio of lung tissue were observed. The expression of lung phosphorylated mitogen-activated protein kinase kinase (p-MEK) and phosphorylated extracellular regulated protein kinases (p-ERK) were detected by Western Blot. Compared with the sham group, the lung histopathological injury was significantly aggravated, and the histopathological injury score was significantly increased, the lung W/D ratio, and total protein content, NE, TNF-α, IL-6, IL-10 in BALF, and p-MEK and p-ERK expressions were significantly increased in LPS group at 6 hours after model setup [the lung histopathological injury score: 0.70±0.04 vs. 0.14±0.13, W/D ratio: 4.79±0.15 vs. 4.35±0.17, protein content (g/L): 1.51±0.36 vs. 0.46±0.13, NE (ng/L): 85.02±11.28 vs. 47.18±10.30, TNF-α (ng/L): 186.61±21.93 vs. 9.18±2.86, IL-6 (ng/L): 193.45±26.54 vs. 13.58±2.54, IL-10 (ng/L): 113.46±31.23 vs. 25.66±9.41, p-MEK/β-actin: 0.246±0.019 vs. 0.178±0.030, p-ERK/β-actin: 0.257±0.013 vs. 0.175±0.014, all P < 0.05], and increase with time after model setup. Compared with the LPS group, BRL-44408 maleate pretreatment for 6 hours could significantly improve the degree of lung injury and reduce the lung histopathological injury score (0.61±0.05 vs. 0.70±0.04), reduce lung W/D weight ratio (4.51±0.22 vs. 4.79±0.15); the expression of NE, TNF-α, IL-6 in BALF were inhibited [NE (ng/L): 55.77±15.86 vs. 85.02±11.28, TNF-α(ng/L): 54.79±12.68 vs. 186.61±21.93, IL-6 (ng/L): 67.66±20.08 vs. 193.45±26.54], in addition, the up-regulation of p-MEK, p-ERK were significantly inhibited (p-MEK/β-actin: 0.204±0.008 vs. 0.246±0.019, p-ERK/β-actin: 0.186±0.024 vs. 0.257±0.013), with statistically significant differences (all P < 0.05). The protein content and the expression of IL-10 in BALF showed no significant difference. α2A-AR blocker BRL-44408 maleate could alleviate endogenous ALI induced by LPS in mice by inhibiting the MEK/ERK pathway.