Regulatory effect of cytokine-induced neutrophil chemoattractant, epithelial neutrophil-activating peptide 78 and pyrrolidine dithiocarbamate on pulmonary neutrophil aggregation mediated by nuclear factor-κB in lipopolysaccharide-induced acute respiratory distress syndrome mice

Abstract

In the present study, the regulatory effect of cytokine-induced neutrophil chemoattractant (CINC) and epithelial neutrophil-activating peptide 78 (ENA-78) on pulmonary neutrophil (PMN) accumulation in lipopolysaccharide (LPS)-induced acute respiratory distress syndrome (ARDS) mice, and the therapeutic effect of pyrrolidine dithiocarbamate (PDTC), was investigated. BALB/c mice were divided into control, LPS and PDTC + LPS groups using a random number table. The phosphorylation of nuclear factor-κB (NF-κB) was detected using a western blot, and the mRNA expression levels of CINC were evaluated using reverse transcription-quantitative polymerase chain reaction. The expression of NF-κB, CINC and ENA-78 was detected using immunohistochemistry. The production of interleukin (IL)-8 and IL-10 in serum and broncho-alveolar lavage fluid (BALF) was analyzed using an enzyme-linked immunosorbent assay. The total number of leukocytes and proportion of PMNs in BALF was also determined. Following injection with LPS (20 mg/kg), the expression levels of p-NF-κB, CINC and ENA-78 were increased in lung tissue, and the expression levels of IL-8, IL-10 and the number of PMNs increased in serum and BALF. However, in comparison with the LPS group, the degree of lung injury was reduced in ARDS mice that were treated with PDTC. In addition, the expression level of p-NF-κB and the production of chemokines in lung tissue decreased in ARDS mice that were treated with PDTC, and the number of PMNs in BALF also decreased. In conclusion, the results of the present study suggest that the LPS-induced phosphorylation of NF-κB may result in the synthesis and release of CINC and ENA-78, which induce the accumulation of PMNs in the lung. Therefore, PDTC may be used to reduce the production of chemokines and cytokines, thereby decreasing the activation of PMNs in lung tissue and reducing the damage of lung tissue in ARDS.

Keywords: acute respiratory distress syndrome; cytokine-induced neutrophil chemoattractant; epithelial neutrophil-activating peptide 78; nuclear factor-κB; polymorphonuclear neutrophils.

Figure 1.

General condition (left panel) and lungs (right panel) of mice in the (A) normal saline control, (B) lipopolysaccharide (LPS) and (C) LPS + pyrrolidine dithiocarbamate (PDTC) group (n=12). Mice were treated with 20 mg/kg LPS with PDTC (0, 40, 120 or 160 mg/kg, intraperitoneally).

Figure 2.

Effect of pyrrolidine dithiocarbamate (PDTC) on lung histopathological changes of mice at 12 and 24 h following intraperitoneal injection with saline, and lipopolysaccharide (LPS) with or without PDTC (stain, hematoxylin and eosin; magnification, ×400). (A) Normal saline group with integrated lung tissue and no inflammatory cells; (B) LPS group at 12 h; (C) LPS group at 24 h with widened lung intervals, highly congested pulmonary interstitia, fractured alveolar walls and numerous infiltrative inflammatory cells; and (D) PDTC + LPS group with milder lung tissue injury in comparison with the LPS group.

Figure 3.

Effect of P on P(A-a)O₂ in the saline, L and P + L group. P (120 mg/kg, intraperitoneally) was administered 30 min prior to L administration (20 mg/kg intraperitoneally). Mice were anesthetized at 2, 6, 12 and 24 h following treatement with NS or L. Blood was collected immediately in order to determine P(A-a)O₂. NS, normal saline; P(A-a)O₂, oxygen partial pressure difference between alveolar and arterial; P, pyrrolidine dithiocarbamate; L, lipopolysaccharide.

Figure 4.

Effect of PDTC on p-NF-κB and NF-κB P65 protein expression in lung tissue and the cytoplasm. (A and B) The expression of p-NF-κB was significantly increased in the LPS group (P<0.01) and significantly decreased in the PDTC + LPS group (P<0.05), demonstrating that PDTC can inhibit the activation of NF-κB. (C and D) NF-κB P65 protein expression was reduced in the cytoplasm of the LPS group (P<0.01); the production of P65 was significantly increased in nuclei of the LPS group in comparison with the NS group (P<0.01). (E and F) P65 expression was significantly increased in the cytoplasm (P<0.05), and significantly decreased in the nucleus, of the PDTC + LPS group in comparison with the LPS group (P<0.01). PDTC, pyrrolidine dithiocarbamate; NF-κB, nuclear factor-κB, p-NF-κB, phosphorylated-NF-κB; LPS, lipopolysaccharide; NS, normal saline; h, hours.

Figure 5.

Change of CINC mRNA expression in lung tissue detected by qPCR. (A) CINC expression was detected by RT-PCR. (B) mRNA expression of CINC was detected by qPCR. **P<0.01 vs. NS group; ^##P<0.01 vs. L group. CINC, cytokine-induced neutrophil chemoattractant; NS, normal saline; L, lipopolysaccharide; P, pyrrolidine dithiocarbamate; qPCR, quantitative polymerase chain reaction.

Figure 6.

Expression of nuclear factor (NF)-κB in lung tissue determined by immunohistochemistry (stain, hematoxylin and eosin; magnification, ×400). (A) In the normal saline group, the positive NF-κB cells were not observed; (B) in the 2 h lipopolysaccharide (LPS) group, low expression levels of NF-κB were detected; (C) in the 12 h LPS group, the appearance of NF-κB positive cells was enhanced in comparison with the 2 h LPS group; and (D) in the 12 h pyrrolidine dithiocarbamate + LPS group, NF-κB expression was reduced in comparison with the 12 h LPS group.

Figure 7.

Cytokine-induced neutrophil chemoattractant (CINC) expression in the lung tissue of mice detected by immunohistochemistry (stain, hematoxylin and eosin; magnification, ×400). (A) Normal saline group, (B) 2 h lipopolysaccharide (LPS) group, (C) 12 h LPS group and (D) 12 h pyrrolidine dithiocarbamate (PDTC) + LPS group. The expression of CINC was aggravatingly expressed in lung tissue of mice with LPS-induced acute respiratory distress syndrome. The number of positive cells stained with CINC in the PDTC + LPS group was markedly decreased in comparison with the 12 h LPS group.

Figure 8.

Expression of epithelial-derived neutrophil-activating protein 78 (ENA-78) in lung tissue, detected using immunohistochemstry (stain, hematoxylin and eosin; magnification, ×400). (A) Normal saline (NS), (B) 2 h lipopolysaccharide (LPS), (C) 12 h LPS and (D) 12 h pyrrolidine dithiocarbamate + LPS group. No ENA-78 positive cells were detected in the NS group. ENA-78 expression was increased in the PDTC + LPS group at 12 h, as compared with the NS group, and decreased in comparison with the LPS group.

Figure 9.

Expression of IL-8 in (A) serum and (B) BALF, and the expression of IL-10 in (C) serum and (D) BALF. *P<0.05, **P<0.01 vs. the NS group; ^#P<0.05, ^##P<0.01, ^###P>0.05 vs. the L group. IL-8, interleukin-8; BALF, bronchoalveolar lavage fluid; NS, normal saline; L, lipopolysaccharide; P, pyrrolidine dithiocarbamate.

Figure 10.

Number of (A) leukocytes and the proportion of (B) PMNs in the BALF of different groups. **P<0.01 vs. the NS group; ^#P<0.05, ^##P<0.01 vs. the L group. PMN, polymorphonuclear; BALF, bronchoalveolar lavage fluid; NS, normal saline; L, lipopolysaccharide; P, pyrrolidine dithiocarbamate; WBC, white blood cell.