Department of Restorative Dentistry and Periodontology, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand.
Department of Family and Community Dentistry, Faculty of Dentistry, Chiang Mai University, Chiang Mai, Thailand.
Int Endod J. 2021 Jun;54(6):951-965. doi: 10.1111/iej.13484. Epub 2021 Feb 19.
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 20 μg mL of Escherichia coli (E. coli) LPS for 24 h, 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 48 h. Production of mtROS was evaluated at 6 and 24 h 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 6 h 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 21 days. 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 24 h (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 24 h. The combination of NAC with MTA promoted mineralization.
研究 N-乙酰半胱氨酸（NAC）、Biodentine、ProRoot MTA 及其组合对脂多糖（LPS）诱导的人牙髓细胞（hDPC）活力、线粒体活性氧（mtROS）产生、矿化以及与炎症细胞因子产生、线粒体动力学和细胞凋亡相关基因表达的影响。
分离的 hDPCs 用 20μg mL 的大肠杆菌（E. coli）LPS 孵育 24 h，除对照组外。将实验分为 8 个实验组：（i）对照组（常规培养基培养的 hDPCs）、（ii）+LPS（整个实验中在 LPS 培养基中培养的 hDPCs）、（iii）-LPS/ 培养基、（iv）-LPS/BD、（v）-LPS/MTA、（vi）-LPS/NAC、（vii）-LPS/BD+NAC 和（viii）-LPS/MTA+NAC。在 24 和 48 h 时使用 Alamar blue 测定细胞活力。在 6 和 24 h 通过 MitoSOX Red 和 MitoTracker Green 评估 mtROS 的产生。在 6 h 使用逆转录聚合酶链反应（RT-PCR）研究 IL-6、TNF-α、Bcl-2、Bax、Mfn-2 和 Drp-1 基因的表达。为了研究分化潜能，将细胞在成骨分化培养基中培养，并在第 14 和 21 天用茜素红染色进行染色。进行 Kruskal-Wallis 检验、Mann-Whitney U 检验和单因素方差分析进行统计分析。
NAC 与 LPS 诱导的 hDPC 活力显著增加相关（P<0.05）。Biodentine 和 MTA 提取物均促进细胞存活，而 NAC 与这些材料提取物的组合在 24 h 时显著增加了存活细胞的数量（P<0.05）。Biodentine、MTA 或 NAC 均未改变 mtROS 水平（P>0.05）。NAC 补充到 MTA 提取物中可显著降低 IL-6 和 TNF-α的表达（P<0.05）。关于线粒体动力学，单独使用 NAC 可促进 Mfn-2/Drp-1 表达的显著增加（P<0.05）。大多数组的 Bcl-2/Bax 基因表达水平与对照组相似。除 LPS 组外，大多数组的矿化产物增加（P<0.05）。
在体外 DPC 中，NAC 的抗氧化作用在 LPS 诱导条件下不明显。NAC 与 Biodentine 或 MTA 联合使用可提高 LPS 诱导的 hDPCs 在 24 h 时的存活率。NAC 与 MTA 的组合促进了矿化。
