Expression and significance of Toll-like receptor-4 in rats lung established by passive smoking or associated with intratracheal instillation of lipopolysaccharide

Abstract

To explore the expression and effect of Toll-like receptor 4 (TLR-4) in the lung tissue of rats established by passive smoking or intratracheal instillation of lipopolysaccharide (LPS). Eight-week-old male Sprague-Dawley rats (n = 15) were randomly divided into 3 groups, including: (1) group A: conventional breeding; (2) group B: the rats were placed into a 120-L organic glass box with twice-daily exposure to cigarette smoking plus an intratracheal instillation of water at Day 1 and 14; (3) Group C: exposure to cigarette smoking the same as group B plus intratracheal instillation of lipopolysaccharide (1 mg/kg) at Day 1 and 14. Four weeks later, general status, arterial blood gas, pulmonary function and histopathology were analyzed. The expressions of TLR-4 and nuclear factor kappa-B (NF-κB) were determined by immunohistochemistry. Western blot was used to measure the protein contents of TLR-4, NF-κB, p-Iκ-Kα/β, Iκ-Kα/β, IκB-α. And real-time polymerase chain reaction (PCR) was employed to detect the mRNA levels of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6). Rats in Groups B and C were marantic with intermittent cough and dyspnea. Peak expiratory flow (PEF) and 50% expiratory flow-volume (EP50) were much lower in Group C ((10.6 ± 1.4), (0.77 ± 0.14) ml/s) than that in Groups A ((13.5 ± 2.0), (1.01 ± 0.08) and B (12.3 ± 0.9), (0.91 ± 0.10) ml/s) (all P < 0.05). Accumulated volume (AV) and carbon dioxide pressure (PCO2) were much higher in Groups B ((4358 ± 1501) ml, (52.77 ± 1.97) mm Hg) (1 mm Hg = 0.133 kPa) and C ((10 077 ± 1866) ml, (51.03 ± 4.96) mm Hg) than that in Group A ((1735 ± 798) ml, (39.57 ± 1.43) mm Hg) (all P < 0.05). Hematoxylin and eosin stain showed chronic bronchitis and emphysema in Groups B and C. Besides, quantitative analysis demonstrated that in unit area, mean lining interval (MLI) and destruction index (DI) in Group B ((84 ± 13) µm, 0.228 ± 0.047) and Group C ((86 ± 10) µm, 0.294 ± 0.060) significantly increased versus Group A ((65 ± 6) µm, 0.036 ± 0.012) (all P < 0.05). Immunohistochemical staining indicated that the expression of TLR-4 in cytoplasm and cytomembrane and NF-κB in nucleus markedly increased in Groups B and C versus Group A. Relative expressions of TLR-4 and NF-κB assayed by Western blot increased in Group B (0.68 ± 0.03, 0.21 ± 0.08) and Group C (1.12 ± 0.11, 0.59 ± 0.06) than that in Group A (1.36 ± 0.07, 1.04 ± 0.08). Compared with Group A, the expression levels of TLR-4, NF-κB and IκB-α and the phosphorylation levels of Iκ-Kα/β in Group B and C significantly increased (all P < 0.05). The mRNA levels of TNF-α and IL-6 increased in Group B (3.95 ± 0.29, 5.04 ± 0.28) and C (5.33 ± 0.26, 7.23 ± 0.39) versus that in Group C (1.00 ± 0.37, 1.00 ± 0.25) (all P < 0.05). Both passive smoking and intratracheal instillation of LPS may cause lung injury analogous to chronic obstructive pulmonary disease via NF-κB signaling pathway. And TLR4 plays an important role in this process.