VAS-2870

Particulate matter 2.5 (PM2.5)-induced oxidative stress has been extensively proposed as a pivotal event in lung diseases. Receptor for advanced glycation end-products (RAGE) is a receptor of pro-inflammatory ligands that has been supported to be implied in the progression of multiple lung diseases. This study attempts to delineate the specific effects of PM2.5 on human bronchial epithelial 16HBE cells in vitro and figure out whether PM2.5 functions via mediating oxidative stress and RAGE. In PM2.5-challenged 16HBE cells, MTT assay detected cell viability. ELISA estimated inflammatory levels. Flow cytometry analysis measured ROS activity and related assay kits examined oxidative stress levels. Western blot tested nuclear factor E2-related factor 2 (Nrf2), RAGE, β-catenin, and mucin 5AC (MUC5AC) expression. Immunofluorescence staining evaluated nuclear translocation of β-catenin. It was noticed that PM2.5 exposure exacerbated inflammatory response, oxidative stress, and mucus production. Additionally, PM2.5 elevated RAGE expression while declined Nrf2 expression as well as stimulated the nuclear translocation of β-catenin. Furthermore, RAGE inhibition or nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor VAS2870 mitigated inflammatory response, oxidative stress, and mucus generation in PM2.5-exposed 16HBE cells. In addition, RAGE inhibition or VAS2870 raised Nrf2 expression, reduced RAGE expression, and hampered β-catenin nuclear translocation. Briefly, PM2.5 might act as a leading driver of inflammatory response and mucus production in lung injury, the mechanism of which might be related to the activation of oxidative stress and the up-regulation of RAGE.

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic G_i protein-coupled P₂Y₁₃ receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P₂Y₁₃ receptors (MRS 2211), Ca²⁺ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca²⁺ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca²⁺ with subsequent elevation of ROS levels. The elevation of [Ca²⁺]_in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of G_i protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P₂Y₁₃ receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P₂Y₁₃ receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P₂Y₁₃ receptor/G_i protein/Ca²⁺/protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner.