Effects of N-acetyl cysteine on mitochondrial ROS, mitochondrial dynamics, and inflammation on lipopolysaccharide-treated human apical papilla cells.

Abstract

N-Acetyl cysteine (NAC), a well-known antioxidant molecule, has been used to modulate oxidative stress and inflammation. However, no studies have examined the effect of NAC in regenerative endodontic procedures (REPs). Therefore, the aim of this study was to investigate the effects of NAC on cell survival, mitochondrial reactive oxygen species (mtROS) production, and inflammatory and mitochondria-related gene expression on lipopolysaccharide (LPS)-treated apical papilla cells (APCs). To assess the NAC concentration, 5 and 10mM NAC were administered to LPS-treated APCs. Cell proliferation was measured at 24, 48, and 72h by using AlamarBlue® assay. The 5-mM concentration was further analyzed using different treatment durations: 10min, 24h, and the entire study period. The mtROS production was quantified using MitoSOX™ Red and MitoTracker™ Green. RT-PCR was used to detect the expression of IL-6 and TNF-α inflammatory genes and mitochondrial morphology-related genes (Mfn-2/Drp-1 and Bcl-2/Bax) at 6 and 24h. The statistical significance level was set at 0.05. Five-millimolar NAC promoted the highest LPS-treated APC proliferation. The use of 24-h NAC stimulated cell proliferation, whereas the entire-period NAC application (> 48h) significantly reduced the cell number. The mtROS levels were slightly altered after NAC induction. Ten-minute NAC treatment downregulated the IL-6 and TNF-α expression, whereas the expression of Bcl-2/Bax and Mfn-2/Drp-1 ratios was upregulated at 6h. Under the LPS-induced inflammatory condition, NAC stimulated APC survival and decreased inflammation. Ten-minute NAC treatment was sufficient to reduce the level of inflammation and maintain the mitochondrial dynamics. Ten-minute NAC application is sufficient to reduce the level of inflammation and maintain the mitochondrial dynamics. Therefore, NAC may be considered as a potential adjunctive irrigation solution in REPs.