Propyl gallate induces cell death in human pulmonary fibroblast through increasing reactive oxygen species levels and depleting glutathione

Abstract

Abstract Background Propyl gallate (PG) has demonstrated anti-growth effects on various cell types. This study aimed to investigate the impact of PG on reactive oxygen species (ROS) and glutathione (GSH) levels in primary human pulmonary fibroblast (HPF) cells. Additionally, the study explored the effects of N-acetyl cysteine (NAC), L-buthionine sulfoximine (BSO), and small interfering RNA (siRNAs) targeting antioxidant genes on ROS, GSH levels, and cell death in PG-treated HPF cells. Methods and Results Intracellular ROS and GSH levels were measured using different fluorescent probe dyes (H2DCFDA, DHE, MitoSOX™ Red and CMFDA) through flow cytometer analysis. PG treatment at concentrations ranging from 100 to 800 µM resulted in increased levels of total ROS and O2•− at early time points (30–180 min and 24 h). Higher concentrations of PG (800–1600 µM) led to an increased number of cells depleted of GSH at 24 h and reduced GSH levels at 30–180 min. PG treatment downregulated the activity of superoxide dismutase (SOD) and upregulated the activity of catalase in HPF cells. Treatment with 800 µM PG increased the number of apoptotic cells and cells with loss of mitochondrial membrane potential (MMP; ∆Ψm) as assessed by Annexin V-FITC/PI and rhodamine 123 staining, respectively. NAC treatment mitigated PG-induced cell death and loss of MMP in HPF cells, accompanied by a decrease in GSH depletion. Conversely, BSO exacerbated cell death and MMP loss induced by PG, independent of ROS and GSH depletion levels. Following transfection with siRNA, the HPF cells were exposed to PG treatment. The siRNAs against SOD1, SOD2, and catalase successfully attenuated cell death in PG-treated HPF cells. In contrast, siRNA against GSH peroxidase enhanced cell death. Conclusions PG induced cell death in HPF cells by increasing ROS levels and depleting GSH. NAC was found to decrease HPF cell death induced by PG, while BSO enhanced cell death. The findings suggest that modulating the antioxidant defense system can influence the cytotoxic effects of PG in HPF cells.