Nrf-2/HO-1 activation protects against oxidative stress and inflammation induced by metal welding fume UFPs in 16HBE cells

Abstract

As one of the main occupational hazards, welding fumes can cause oxidative damage and induce series of diseases, such as COPD or asthma. To clarify the effects of the metal fume ultrafine particulates (MF-UFPs) of welding fumes on oxidative damage, UFPs were collected by melt inert gas (MIG) and manual metal arc (MMA) welding, and the composition was confirmed. Human bronchial epithelial 16HBE cells were treated with 0-1000 µg/cm² MF-UFPs to analyse the cytotoxicity, oxidative stress and cytokines. The protein and mRNA expression of Keap1-Nrf-2/antioxidant response elements (AREs) signalling pathway components were also analysed. After 4 h of treatment, the cell viability decreased 25% after 33.85 and 32.81 µg/cm² MIG/MMA-UFPs treated. The intracellular ATP concentrations were also decreased significantly, while LDH leakage was increased. The decreased mitochondrial membrane potential and increased ROS suggested the occurrence of oxidative damage, and the results of proteome profiling arrays also showed a significant increase in IL-6 and IL-8. The expression of AREs which related to antioxidant and anti-inflammatory were also increased. These results indicate that the MF-UFPs can cause oxidative stress in 16HBE cells and activate the Nrf-2/ARE signalling pathway to against oxidative damage.

Keywords: Nrf-2/ARE; Oxidative stress; Ultrafine particles; Welding fume.

Fig. 1

The cytotoxicity of MF-UFPs. (a) Cell viability, (b) BMD prediction model, (c) ATP levels and (d) LDH leakage rate after treated with MIG and MMA. *: p < 0.05; Blue line: Estimated probability line between cell viability and treatment dose; Light green line: The response value of cell viability at BMD; Red circle: The cell viability and treatment dose data; Green line: The BMD value; Yellow line: The BMDL value.

Fig. 2

MF-UFPs reduced MMP and increased ROS and CAT activity in 16HBE cells. (a) Mitochondrial membrane potential after JC-1 staining. (b and c) ROS level detected by DCFH-DA. (d) Changes in catalase activity after treatment. Scale bar = 100 nm (* p < 0.05).

Fig. 3

The expression levels of cytokines after MF-UFPs treatment. Total protein was collected from MIG- and MMA-treated cells under 33.85 and 32.81 µg/cm² treatment conditions, respectively. Each array membrane was incubated with 200 µg of sample at 4 °C overnight. The red box highlights the upregulated expression level of the detected plots.

Fig. 4

The protein expression levels of Nrf-2, Keap1, HO-1, NQO-1, CAT and GAPDH after exposure with MF-UFPs. (a) The area under the curve of protein and (b) Western blot at 4 h after treatment with MIG and MMA (* p < 0.05).

Fig. 5

Heatmap of Nrf-2/ARE pathway gene expression levels. The heatmap shows the relative expression levels of the AREs; The CV75 condition was 33.85 µg/cm² for MIG and 32.81 µg/cm² for MMA; The CV50 treatment condition was 151.42 µg/cm² for MIG and 142.12 µg/cm² for MMA. red indicates upregulated genes, and blue indicates downregulated genes.