Regulatory mechanism of pyrrolidine dithiocarbamate is mediated by nuclear factor-κB and inhibits neutrophil accumulation in ARDS mice

Abstract

The aim of the present study was to investigate the regulatory mechanism of nuclear factor (NF)-κB on polymorphonuclear neutrophil (PMN) accumulation and the inflammatory response in lung tissues with acute respiratory distress syndrome (ARDS), as well as the therapeutic effect of pyrrolidine dithiocarbamate (PDTC). Mouse models of ARDS were established by intraperitoneal injection of lipopolysaccharide (LPS). BALB/c mice were divided into control, LPS and PDTC + LPS groups. The expression of PMN adhesion molecules, CD11b/CD18 and intercellular adhesion molecule-1 (ICAM-1), were detected by immunohistochemistry, while the protein expression levels of NF-κB p65 in the lung tissue were analyzed by western blot analysis. In addition, flow cytometry was used to investigate the apoptosis rate of PMNs in the bronchoalveolar fluid, and the expression levels of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α and myeloperoxidase (MPO) activity were also determined. Following an intraperitoneal injection of LPS, alveolar septum rupture, pulmonary interstitial hyperemia and PMN infiltration in the alveolar was observed. The protein expression of p65 in the pulmonary cytoplasm decreased, while the expression of p65 in the nucleus increased. The levels of IL-8, IL-1β and TNF-α increased and the high expression status was maintained for 24 h. As the time increased, CD11b/CD18 and ICAM-1 expression increased, as well as MPO activity, while the apoptosis of PMNs was delayed. Compared with the LPS group, the expression of p65 in the pulmonary cytoplasm and the PMN apoptosis rate increased following PDTC intervention, while the expression of p65 in the nucleus decreased, as well as the expression levels of the cytokines and MPO activity. Therefore, PDTC reduced the production of inflammatory cytokines via the NF-κB pathway, which reduced the activation of PMNs in the lung tissue and promoted PMN apoptosis.

Keywords: CD11b/CD18; acute respiratory distress syndrome; intercellular adhesion molecule-1; nuclear factor-κB; pyrrolidine dithiocarbamate.

Figure 1

Effect of PDTC on LPS-induced mortality in mice (n=10). Mice were challenged with 20 mg/kg LPS with or without various doses of PDTC (40, 120 or 160 mg/kg, i.p.). Survival rate was observed after 12, 24, 36, 48, 60 and 72 h. The percentage survival rate of the mice was expressed using Kaplan-Meier survival curves. PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide.

Figure 2

Effect of PDTC on lung histopathological changes at 4 and 24 h following saline and LPS intraperitoneal injection (HE stain; magnification, ×400). (A) Control group; (B) 4 and (C) 24 h LPS groups exhibited pulmonary edema, infiltration of inflammatory cells and alveolar damage; (D) 24 h PDTC + LPS group exhibited significantly alleviated lung injury and less damage compared with the LPS group. PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide; HE, hematoxylin and eosin.

Figure 3

Effect of PDTC on the W/D ratio of lung tissue and protein leakage in BALF samples of LPS-induced ARDS mice. PDTC (120 mg/kg, i.p.) was administered 30 min prior to LPS administration (20 mg/kg, i.p.). Mice were anesthetized at 4, 12 and 24 h following the saline or LPS challenge. Lung tissue samples, BALF and serum were then collected immediately for (A) lung W/D ratio and (B) lung permeability index assays. Values are presented as the mean ± standard deviation. ^*P<0.05 and ^**P<0.01, vs. control; ^#P<0.05, vs. LPS group (n=10). NS, normal saline; PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide; W/D, wet to dry weight; BALF, bronchoalveolar lavage fluid; ARDS, acute respiratory distress syndrome.

Figure 4

Effect of PDTC on the PMN apoptosis rate in BALF samples and the pathology scores of LPS-induced ARDS mice. Mice were anesthetized at 4, 12 and 24 h following the saline or LPS challenge. BALF and lung tissues were then collected immediately for the determination of (A) apoptosis rates and (B) histological scores. Values are presented as the mean ± standard deviation. ^*P<0.05 and ^**P<0.01, vs. control; ^#P<0.05 and ^##P<0.01, vs. LPS group (n=10). NS, normal saline; PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide; BALF, bronchoalveolar lavage fluid; ARDS, acute respiratory distress syndrome; PMN, polymorphonuclear neutrophil.

Figure 5

Effect of PDTC on LPS-induced NF-κB p65 expression in the cytoplasm and nucleus. Representative western blots and quantitative protein expression of NF-κB p65 in the (A and B) cytoplasm and (C and D) nucleus, respectively. Values are presented as the mean ± standard deviation. ^**P<0.01, vs. control; ^#P<0.05 and ^##P<0.01, vs. LPS group (n=10). NS, normal saline; PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide; NF, nuclear factor.

Figure 6

Effect of PDTC on the expression of inflammatory cytokines, including (A) TNF-α, (B) IL-1β and (C) IL-8, in the BALF and the (D) activity of MPO in the lung tissues. Values are presented as the mean ± standard deviation. ^**P<0.01, vs. control; ^#P<0.05 and ^##P<0.01, vs. LPS group (n=10). NS, normal saline; PDTC, pyrrolidine dithiocarbamate; BALF, bronchoalveolar lavage fluid; MPO, myeloperoxidase; TNF, tumor necrosis factor; IL, interleukin.

Figure 7

Effect of PDTC on the expression of CD11b/CD18 in the lung tissues of the (A) control, (B) 4 h LPS, (C) 24 h LPS and (D) 24 h PDTC + LPS groups. Mice were treated with PDTC (120 mg/kg, i.p.) prior to the adminstration of LPS (20 mg/kg, i.p.). Mice were anesthetized and lung tissue samples were obtained at 4 and 24 h following the saline or LPS challenge. PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide.

Figure 8

Effect of PDTC on the expression of ICAM-1 in the lung tissues of the (A) control, (B) 4 h LPS, (C) 24 h LPS and (D) 24 h PDTC + LPS groups. Mice were treated with PDTC (120 mg/kg, i.p.) prior to the administration of LPS (20 mg/kg, i.p.). Mice were anesthetized and lung tissue samples were obtained at 4 and 24 h following the saline or LPS challenge. PDTC, pyrrolidine dithiocarbamate; LPS, lipopolysaccharide; ICAM-1, intercellular adhesion molecule-1.