TF-343 Alleviates Diesel Exhaust Particulate-Induced Lung Inflammation via Modulation of Nuclear Factor- κ B Signaling

Abstract

Inhalation of diesel exhaust particulate (DEP) causes oxidative stress-induced lung inflammation. This study investigated the protective effects of TF-343, an antioxidant and anti-inflammatory agent, in mouse and cellular models of DEP-induced lung inflammation as well as the underlying molecular mechanisms. Mice were intratracheally instilled with DEP or vehicle (0.05% Tween 80 in saline). TF-343 was orally administered for 3 weeks. Cell counts and histological analysis of lung tissue showed that DEP exposure increased the infiltration of neutrophils and macrophages in the peribronchial/perivascular/interstitial regions, with macrophages harboring black pigments observed in alveoli. TF-343 pretreatment reduced lung inflammation caused by DEP exposure. In an in vitro study using alveolar macrophages (AMs), DEP exposure reduced cell viability and increased the levels of intracellular reactive oxygen species and inflammatory genes (IL-1β, inhibitor of nuclear factor- (NF-) κB (IκB), and Toll-like receptor 4), effects that were reduced by TF-343. A western blot analysis showed that the IκB degradation-induced increase in NF-κB nuclear localization caused by DEP was reversed by TF-343. In conclusion, TF-343 reduces DEP-induced lung inflammation by suppressing NF-κB signaling and may protect against adverse respiratory effects caused by DEP exposure.

Figure 1

Diagram of in vivo experimental protocol. DEP: diesel exhaust particulate; i.t: intratracheal injection.

Figure 2

Changes in (a) body weight and (b-d) relative organ weight in DEP- and vehicle-treated mice. Lung (b), spleen (c), and thymus (d) weights were calculated with the following formula: relative organ weight = organ weight (g)/final body weight (g) × 100%. Data represent mean ± SD (n = 5 per group). ^#P < 0.05 vs. vehicle control.

Figure 3

Changes in the cell population of BALF from mice treated with vehicle (VEH) or DEP, alone or in conjunction with TF-343 (200, 400, or 800 mg/kg) or Dexa (1 mg/kg). Data represent mean ± SD (n = 5 per group). ^#P < 0.05 vs. vehicle control, ^∗P < 0.05 vs. DEP+VEH.

Figure 4

Histological changes in lung tissue caused by DEP treatment. Representative H&E-stained sections of the lung obtained from mice treated with (a) vehicle or (b-f) DEP alone (b) or in conjunction with TF-343 at 200 mg/kg (c), 400 mg/kg (d), or 800 mg/kg (e) or Dexa (1 mg/kg) (f). Red and black arrows indicate black particle-laden alveolar macrophages and inflammatory cells, respectively. Scale bars, 20 μm.

Figure 5

Intracellular ROS levels and viability of DEP-treated AMs. (a) ROS levels were measured by DCF-DA staining. Data represent mean ± SD (n = 3 per group). ^#P < 0.05 vs. control. (b) Evaluation of cytotoxicity with the MTT assay. Data represent mean ± SD (n = 8 per group). ^##P < 0.01 vs. control, ^∗P < 0.05 vs. DEP-treated group. AMs were pretreated overnight with TF-343 (100 or 200 μg/ml) before adding DEP (100 μg/ml). At 3 h after DEP stimulation, DCF-DA staining and the MTT assay were performed.

Figure 6

Expression levels of inflammation-related genes including (a) IL-1β, (b) IκB, and (c) TLR4 in DEP-stimulated AMs. AMs were pretreated overnight with TF-343 (100 or 200 μg/ml) before adding DEP (100 μg/ml). Real-time PCR analysis was performed 48 h after DEP stimulation. Data represent mean ± SD (n = 5 per group). ^##P < 0.01 vs. control, ^∗P < 0.05 vs. DEP-treated group.

Figure 7

(a–c) Representative western blots and (d–f) relative density of IκB and NF-κB in DEP-stimulated AMs. Data represent the mean ± SD (n = 5 per group). ^##P < 0.01 vs. control, ^∗P < 0.05 vs. DEP-treated group.