α1-adrenoceptor stimulation ameliorates lipopolysaccharide-induced lung injury by inhibiting alveolar macrophage inflammatory responses through NF-κB and ERK1/2 pathway in ARDS

Abstract

Introduction: Catecholamines such as norepinephrine or epinephrine have been reported to participate in the development of acute respiratory distress syndrome (ARDS) by activating adrenergic receptors (ARs). But the role of α1-AR in this process has yet to be elucidated.

Methods: In this study, ARDS mouse model was induced by intratracheal instillation of lipopolysaccharide. After treatment with α1-AR agonist phenylephrine or antagonist prazosin, lung pathological injury, alveolar barrier disruption and inflammation, and haemodynamic changes were evaluated. Cytokine levels and cell viability of alveolar macrophages were measured in vitro. Nuclear factor κB (NF-κB), mitogen-activated protein kinase, and Akt signalling pathways were analysed by western blot.

Results: It showed that α1-AR activation alleviated lung injuries, including reduced histopathological damage, cytokine expression, and inflammatory cell infiltration, and improved alveolar capillary barrier integrity of ARDS mice without influencing cardiovascular haemodynamics. In vitro experiments suggested that α1-AR stimulation inhibited secretion of TNF-α, IL-6, CXCL2/MIP-2, and promoted IL-10 secretion, but did not affect cell viability. Moreover, α1-AR stimulation inhibited NF-κB and enhanced ERK1/2 activation without significantly influencing p38, JNK, or Akt activation.

Discussion: Our studies reveal that α1-AR stimulation could ameliorate lipopolysaccharide-induced lung injury by inhibiting NF-κB and promoting ERK1/2 to suppress excessive inflammatory responses of alveolar macrophages.

Keywords: NF-κb; acute respiratory distress syndrome; alveolar macrophage; inflammation; α1 adrenergic receptor.

Figure 1

Phenylephrine (PE) attenuated lung pathological injury and alveolar capillary barrier disruption without influencing the cardiovascular haemodynamics of ARDS mice. Mice were given an intratracheal instillation of 2 mg/kg lipopolysaccharide (LPS) to induce ARDS, and PE was injected into trachea 20 min before LPS stimulation. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected 24 h after LPS stimulation. (A) Method for intratracheal instillation of LPS or PE in mice. (B) Expression of α₁-AR in lung tissues of mice. (C) Haematoxylin and eosin staining of lung slices. Scale bar = 100 μM. (D) Histology scores of lungs were judged according to guidelines of the American Thoracic Society. (E) Wet/dry weight ratio of lung tissues. (F) Levels of total protein in BALF. (G) Levels of albumin in BALF. (H) Heart rate (HR), systolic blood pressure (SP), diastolic blood pressure (DP), and mean artery pressure (MAP) of mice were monitored before establishing ARDS model. (I) HR, SP, DP, and MAP of mice were monitored 24 h after establishing ARDS model. Data are represented as the mean ± SD, n = 6–10 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Phenylephrine (PE) ameliorated lung inflammation in ARDS mice. Mice were given an intratracheal instillation of 2 mg/kg lipopolysaccharide (LPS) to induce ARDS, and PE was injected into trachea 20 min before LPS stimulation. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected 24 h after stimulation with LPS. (A) Inflammatory cells in BALF stained by Wright-Giemsa stain. Scale bar = 100 μM. (B) Counts of total inflammatory cells in BALF. (C) Counts of neutrophils in BALF. Levels of TNF-α (D), IL-6 (E), and CXCL2/MIP-2 (F) in BALF. Data are represented as the mean ± SD, n = 6–10 per group. *p < 0.05, **p < 0.01.

Figure 3

Phenylephrine (PE) inhibited inflammation in lipopolysaccharide (LPS)-activated murine alveolar macrophages without influencing cell viability. MH-S cells were incubated with PE (10^-8-10^-5 M) for 30 min, followed by LPS (100 ng/mL) for 6 h Expression of α₁-AR in MH-S cells was detected by immunofluorescence (A) and western blotting (B). Levels of TNF-α (C), IL-6 (D), CXCL2/MIP-2 (E), and IL-10 (F) released from MH-S cells. (G) Live and dead cells were stained by calcein-AM and propidium iodide, respectively. Scale bar = 200 μM. (H) Cell viability was measured by CCK-8 assay. Data are represented as the mean ± SD, n = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Phenylephrine (PE) suppressed NF-κB activation and enhanced ERK1/2 activation in alveolar macrophages stimulated by lipopolysaccharide (LPS). MH-S cells were incubated with PE (10^-8-10^-5 M) for 30 min, followed by LPS (100 ng/mL) for 30 min. (A) Western blotting was used to evaluate activation of p65, Akt, p38, ERK1/2, and JNK. Phosphorylation of p65 (B), Akt (C), p38 (D), ERK1/2 (E), and JNK (F) were analysed according to grey values. Data are represented as the mean ± SD, n = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Prazosin (PRA) reversed the effect of phenylephrine (PE) on lung pathological injury and alveolar capillary barrier disruption without influencing the cardiovascular haemodynamic of ARDS mice. Mice were given an intratracheal instillation of 2 mg/kg lipopolysaccharide (LPS) to induce ARDS. PRA and PE were also injected into the trachea 40 min or 20 min before LPS stimulation. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected 24 h after LPS stimulation. (A) Haematoxylin and eosin staining of lung slices. Scale bar = 100 μM. (B) Histology scores of lungs were judged according to guidelines of the American Thoracic Society. (C) Wet/dry weight ratio of lung tissues. (D) Levels of total protein in BALF. (E) Levels of albumin in BALF. (F) Heart rate (HR), systolic blood pressure (SP), diastolic blood pressure (DP), and me an artery pressure (MAP) of mice were monitored before establishing ARDS model. (G) HR, SP, DP, and MAP of mice were monitored 24 h after establishing ARDS model. Data are represented as the mean ± SD, n = 6–9 per group. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Prazosin (PRA) reversed the effect of phenylephrine (PE) on lung inflammation in ARDS mice. Mice were given an intratracheal instillation of 2 mg/kg lipopolysaccharide (LPS) to induce ARDS. PRA (10^-5 M) and PE (10^-5 M) were also injected into the trachea 40 min or 20 min before LPS stimulation. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected 24 h after LPS stimulation. (A) Inflammatory cells in BALF stained by Wright-Giemsa stain. Scale bar = 100 μM. (B) Counts of total inflammatory cells in BALF. (C) Counts of neutrophils in BALF. Levels of TNF-α (D), IL-6 (E), and CXCL2/MIP-2 (F) in BALF. Data are represented as the mean ± SD, n = 6–10 per group. *p < 0.05, **p < 0.01.

Figure 7

Prazosin (PRA) reversed the effect of phenylephrine (PE) on inflammation in lipopolysaccharide (LPS)-activated murine alveolar macrophages without influencing cell viability by suppressing NF-κB activation and enhancing ERK1/2 activation. MH-S cells were incubated with PRA (10^-5 M) for 30 min, followed by PE (10^-5 M) treatment for 30 min, and then LPS (100 ng/mL) for 6 h Levels of TNF-α (A), IL-6 (B), CXCL2/MIP-2 (C), and IL-10 (D) released from MH-S cells. (E) Live and dead cells were stained by calcein-AM and propidium iodide, respectively. Scale bar = 200 μM. (F) Cell viability was measured by CCK-8 assay. (G) Western blotting was used to evaluate activation of p65 and ERK1/2. (H) Phosphorylation of p65 and ERK1/2 were analysed according to grey values. Data are represented as the mean ± SD, n = 5 per group. *p < 0.05, **p < 0.01, ***p < 0.001.