Alterations of mitochondrial dynamics, inflammation and mineralization potential of lipopolysaccharide-induced human dental pulp cells after exposure to N-acetyl cysteine, Biodentine or ProRoot MTA.

Abstract

To investigate the effects of N-acetyl cysteine (NAC), Biodentine, ProRoot MTA and their combinations, on cell viability, mitochondrial reactive oxygen species (mtROS) production, mineralization and on the expression of genes related to inflammatory cytokine production, mitochondrial dynamics and cell apoptosis of lipopolysaccharide (LPS)-induced human dental pulp cells (hDPCs). Isolated hDPCs were exposed to 20μgmL-1 of Escherichia coli (E. coli) LPS for 24h, before the experiment, except for the control group. Eight experimental groups were assigned: (i) control (hDPCs cultured in regular medium), (ii) +LPS (hDPCs cultured in LPS medium throughout the experiment), (iii) -LPS/Media, (iv) -LPS/BD, (v) -LPS/MTA, (vi) -LPS/NAC, (vii) -LPS/BD+NAC and (viii) -LPS/MTA+NAC. Cell viability was measured using Alamar blue assay at 24 and 48h. Production of mtROS was evaluated at 6 and 24h by MitoSOX Red and MitoTracker Green. The expressions of IL-6, TNF-α, Bcl-2, Bax, Mfn-2 and Drp-1 genes were investigated at 6h using reverse transcriptase-polymerase chain reaction (RT-PCR). For differentiation potential, cells were cultured in the osteogenic differentiation media and stained using Alizarin red assay at 14 and 21days. The Kruskal-Wallis test, Mann-Whitney U test and one-way anova were performed for statistical analysis. NAC was associated with significantly greater LPS-induced hDPC viability (P<0.05). Both Biodentine and MTA extracts promoted cell survival, whereas the combination of NAC to these material extracts significantly increased the number of viable cells at 24h (P<0.05). Biodentine, MTA or NAC did not alter the mtROS level (P>0.05). NAC supplementation to the MTA extract significantly reduced the level of IL-6 and TNF-α expression (P<0.05). Regarding mitochondrial dynamics, the use of NAC alone promoted significant Mfn-2/Drp-1 expression (P<0.05). Most of the groups exhibited a level of Bcl-2/Bax gene expression similar to that of the control group. The increases in mineralization productions were observed in most of the groups, except the LPS group (P<0.05). The antioxidant effect of NAC was not evident under the LPS-induced condition in DPC in vitro. NAC combined either with Biodentine or MTA improved LPS-induced hDPCs survival at 24h. The combination of NAC with MTA promoted mineralization.