Effect of TLR4/MyD88 signaling pathway on sepsis-associated acute respiratory distress syndrome in rats, via regulation of macrophage activation and inflammatory response

Abstract

The present study aimed to investigate the effects of the Toll-like receptor (TLR)4/myeloid differentiation primary response (MyD)88 signaling pathway on sepsis-associated acute respiratory distress syndrome (ARDS) in rats, and the involvement of macrophage activation and the inflammatory response. A total of 36 specific pathogen-free male Sprague-Dawley rats were selected to establish the rat model of sepsis-associated ARDS using cecal ligation and puncture (CLP). Rats were assigned into the Ab (anti-TLR4 monoclonal antibody)-CLP, CLP and Sham groups. Arterial partial pressure of oxygen (P_aO₂) was detected using blood gas analysis. Bronchoalveolar lavage fluid (BALF) and alveolar macrophages were collected. The pathological structure of lung tissue was observed following hematoxylin-eosin staining. The ultrastructural alterations of alveolar epithelial cells were observed under transmission electron microscope. The ratios of wet/dry weight of lung tissue and total protein content in BALF were measured. The concentration of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in BALF and peripheral blood was determined by enzyme-linked immunosorbent assay. The TLR4, TLR9, MyD88 and nuclear factor (NF)-κΒ mRNA and protein expression levels in alveolar macrophages were measured by reverse transcription-quantitative polymerase chain reaction and western blotting. Compared with the Sham group, the rats in the CLP group demonstrated significantly increased respiratory frequency, lung permeability, lung edema, inflammatory infiltration, TNF-α and IL-1β expression levels in BALF and peripheral blood and TLR4, TLR9, MyD88 and NF-κΒ expression levels in macrophages, however decreased arterial P_aO₂. Following pretreatment with anti-TLR4 monoclonal antibody, rats exhibited decreased lung injury, inflammatory infiltration, lung edema, TNF-α and IL-1β expressions in BALF and peripheral blood, and TLR4, TLR9, MyD88 and NF-κΒ expression levels in macrophages, with increased arterial P_aO₂. These results suggested that the inhibition of TLR4/MyD88 signaling pathway may relieve sepsis-associated ARDS in rats through regulating macrophage activation and the inflammatory response.

Keywords: TLR4/MyD88 signaling pathway; acute respiratory distress syndrome; inflammatory response; macrophage activation; sepsis.

Figure 1.

Respiratory frequency and arterial P_aO₂ of rats in three groups. (A) Comparison of respiratory frequency of rats among the Sham, CLP and Ab-CLP groups; (B) comparison of the arterial P_aO₂ of rats among the Sham, CLP and Ab-CLP groups by blood gas analysis; **P<0.005 compared with the CLP group. P_aO₂, partial pressure of oxygen; CLP, cecal ligation puncture; Ab, anti-TLR4 monoclonal antibody.

Figure 2.

Pathological structure of lung tissue of rats by H&E staining in three groups (magnification, ×200). H&E, hematoxylin and eosin; CLP, cecal ligation puncture; Ab, anti-TLR4 monoclonal antibody.

Figure 3.

Cellular structure of type II alveolar epithelial cells of rats in three groups under transmmision electron microscope (magnification, ×2,800). CLP, cecal ligation puncture; Ab, anti-TLR4 monoclonal antibody; Lb, Lamellar bodies; Mv, Microvilli; Nu, Nucleus.

Figure 4.

Comparisons of W/D ratios and total protein content in BALF of rats in three groups. (A) Comparison of the W/D ratios of rats among the Sham, CLP and Ab-CLP groups; (B) comparison of the total protein content in BALF of rats among the Sham, CLP and Ab-CLP groups; **P<0.005 compared with the CLP group. W/D, wet weight/dry weight; BALF, bronchoalveolar lavage fluid; CLP, cecal ligation puncture; Ab, anti-TLR4 monoclonal antibody.

Figure 5.

Comparison of the protein contents of inflammatory factors c in BALF and peripheral blood among the three groups. (A) Comparison of the protein contents of inflammatory factors TNF-α and IL-1β in BALF among the Sham, CLP and Ab-CLP groups; (B) comparison of the protein contents of inflammatory factors TNF-α and IL-1β in peripheral blood among the Sham, CLP and Ab-CLP groups; **P<0.005 compared with the CLP group; ***P<0.001 compared with the CLP group. BALF, bronchoalveolar lavage fluid; CLP, cecal ligation puncture; Ab, anti-TLR4 monoclonal antibody; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β.

Figure 6.

Images of alveolar macrophage after wright-giemsa staining and trypan blue staining (magnification, ×400). (A) Image of alveolar macrophage after wright-giemsa staining; (B) image of alveolar macrophage after trypan blue staining.

Figure 7.

Comparison of the mRNA and protein expressions of TLR4/MyD88 signaling pathway-related factors in alveolar macrophage among the three groups. (A) Comparison of TLR4, TLR9, MyD88 and NF-κΒ P65 mRNA expressions in alveolar macrophage of rats among the Sham, CLP and Ab-CLP groups; (B) protein bands of TLR4, TLR9, MyD88 and NF-κΒ P65 in the Sham, CLP and Ab-CLP detected by western blotting; (C) comparison of TLR4, TLR9, MyD88 and NF-κΒ P65 protein expressions in alveolar macrophage of rats among the Sham, CLP and Ab-CLP groups; **P<0.005 compared with the CLP group; *P<0.05 compared with the CLP group. CLP, cecal ligation puncture; Ab, anti-TLR-4 monoclonal antibody; TLR4, Toll-like receptor 4; TLR9, Toll-like receptor 9; MyD88, myeloid differentiation primary-response protein 88; NF-κB, nuclear factor-κB; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.