Copper oxide nanoparticles exacerbate chronic obstructive pulmonary disease by activating the TXNIP-NLRP3 signaling pathway

Abstract

Background: Although copper oxide nanoparticles (CuONPs) offer certain benefits to humans, they can be toxic to organs and exacerbate underlying diseases upon exposure. Chronic obstructive pulmonary disease (COPD), induced by smoking, can worsen with exposure to various harmful particles. However, the specific impact of CuONPs on COPD and the underlying mechanisms remain unknown. In this study, we investigated the toxic effects of CuONPs on the respiratory tract, the pathophysiology of CuONPs exposure-induced COPD, and the mechanism of CuONPs toxicity, focusing on thioredoxin-interacting protein (TXNIP) signaling using a cigarette smoke condensate (CSC)-induced COPD model.

Results: In the toxicity study, CuONPs exposure induced an inflammatory response in the respiratory tract, including inflammatory cell infiltration, cytokine production, and mucus secretion, which were accompanied by increased TXNIP, NOD-like receptor protein 3 (NLRP3), caspase-1, and interleukin (IL)-1β. In the COPD model, CuONPs exposure induced the elevation of various indexes related to COPD, as well as increased TXNIP expression. Additionally, TNXIP-knockout (KO) mice showed a significantly decreased expression of NLRP3, caspase-1, and IL-1β and inflammatory responses in CuONPs-exposed COPD mice. These results were consistent with the results of an in vitro experiment using H292 cells. By contrast, TNXIP-overexpressed mice had a markedly increased expression of NLRP3, caspase-1, and IL-1β and inflammatory responses in CuONPs-exposed COPD mice.

Conclusions: We elucidated the exacerbating effect of CuONPs exposure on the respiratory tract with underlying COPD, as well as related signaling transduction via TXNIP regulation. CuONPs exposure significantly increased inflammatory responses in the respiratory tract, which was correlated with elevated TXNIP-NLRP3 signaling.

Keywords: Chronic obstructive pulmonary disease; Copper oxide nanoparticles; Inflammation; NOD-like receptor protein 3; Thioredoxin-interacting protein.

Fig. 1

Physicochemical characteristics of CuONPs. (A) The morphology of CuONPs was measured by scanning electric microscopy (Bar = 200 and 100 nm) and transmission electron microscopy (Bar = 200 and 50 nm). (B) The purity of CuONPs was measured by energy-dispersive X ray spectroscopy (Cu: 45.37%, O: 54.63%; Bar = 500 nm)

Fig. 2

Pathophysiological alteration of CuONPs-exposed mice. (A) Expression of TXNIP (× 200, Bar = 60 nm). (B–F) Protein expression and relative densitometric values. Data are indicated as means ± SD (n = 3). ^*, ^**, ^*** Significant differences from the NC group, p < 0.05, p < 0.01, and p < 0.001, respectively

Fig. 3

Pathophysiological alterations of CuONPs-treated NCI-H292 cells. (A) TXNIP and NLRP3 expression by double-immunofluorescence stain (Bar = 10 μm). (B–F) Protein expression and relative densitometric values. Data are indicated as means ± SD (n = 3). ^*, ^**, ^*** Significant differences from the control group, p < 0.05, p < 0.01, and p < 0.001, respectively

Fig. 4

Effects of downregulation of TXNIP on CuONPs-provoked inflammation in NCI-H292 cells. (A–E) Protein expression and relative densitometric values. Data are indicated as means ± SD (n = 3). ^**, ^*** Significant differences from the scrambled siRNA group, p < 0.01 and p < 0.001, respectively; ^#, ^##, ^### Significant differences from the scrambled siRNA + CuONPs group, p < 0.01, p < 0.05, and p < 0.001, respectively

Fig. 5

Effects of CuONPs exposure on the progression of COPD mice. (A–D) Inflammatory cell counts in BALF. (E–G) Inflammatory cytokines in BALF. (H) Staining with hematoxylin and eosin (× 70, Bar = 100 μm) and periodic acid-schiff (× 100, × 400, Bar = 100 μm) on lung tissue. The black arrows represent inflammatory cell infiltration. (I and J) Quantitative analysis of the inflammatory infiltration and mucus production, respectively. (K) 8-OhdG expression by immunofluorescence stain (Bar = 50 μm). (L and M) The levels of MDA and the activities of SOD in lung. Data are indicated as means ± SD (n = 6). ^*, ^**, ^*** Significant differences from the NC group, p < 0.05, p < 0.01, and p < 0.001, respectively; ^#, ^##, ^### Significant differences from the COPD group, p < 0.01, p < 0.05, and p < 0.001, respectively

Fig. 6

Effects of CuONPs exposure on TXNIP related signaling pathway in COPD mice. (A–C) Expression of TXNIP and IL-1β (× 200, Bar = 60 nm) and expression value. (D–H) Protein expression and relative densitometric values. Data are indicated as means ± SD (n = 3). ^*, ^** Significant differences from the NC group, p < 0.05 and p < 0.01, respectively; ^#, ^##, ^### Significant differences from the COPD group, p < 0.01, p < 0.05, and p < 0.001, respectively

Fig. 7

Effects of TXNIP overexpression on pathophysiological alteration in CuONPs-exposed COPD mice. (A–D) Inflammatory cell counts in BALF. (E–G) Inflammatory cytokines in BALF. (I) Staining with hematoxylin and eosin (× 60, Bar = 200 μm) on lung tissue. Black arrows represent inflammatory cell infiltration. (J) Staining with periodic acid-schiff (× 100, Bar = 100 μm) on lung tissue. (H and K) Quantitative analysis of the inflammatory infiltration and mucus production, respectively. (L) 8-OhdG expression by immunofluorescence stain (Bar = 20 μm). (M and N) The levels of MDA and the activities of SOD in lung. (O and Q) Expression of IL-1β (× 200, Bar = 60 nm) and expression value. (P and R–T) Protein expression and relative densitometric values. Data are indicated as means ± SD (n = 6; A–G, n = 3; H–T). Significant differences are shown by the following symbols: ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001

Fig. 8

Effects of TXNIP deletion on the pathophysiological alteration in CuONPs-exposed COPD mice. (A–D) Inflammatory cell counts in BALF. (E–G) Inflammatory cytokines in BALF. (I) Staining with hematoxylin and eosin (× 70, Bar = 100 μm) on lung tissue. Black arrows represent inflammatory cell infiltration. (J) Staining with periodic acid-schiff (× 100, Bar = 100 μm) on lung tissue. (H and K) Quantitative analysis of the inflammatory infiltration and mucus production, respectively. (L) 8-OhdG expression by immunofluorescence stain (Bar = 20 μm). (M and N) The levels of MDA and the activities of SOD in lung. (O and Q) Expression of IL-1β (× 200, Bar = 60 nm) and expression value. (P and R–T) Protein expression and relative densitometric values. Data are indicated as means ± SD (n = 6; A–G, n = 3; H–T). Significant differences are shown by the following symbols: ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001