Introduction

Periodontal healing requires an adequate number of periodontal ligament (PDL) cells to rebuild the impaired tissue. Phenytoin (PHT) has been reported to promote wound healing and extracellular matrix deposition, which indicates its promising application of periodontal healing. However, the effects of PHT on PDL cells behavior and the underlying mechanism are still unknown.

Methods

Human PDL cells were cultured and identified. 20–100 μg/mL PHT were used in our study. The proliferation of PDL cells was determined by the EdU assay. A wound healing assay was used to detect cell migration. Matrix metalloproteinase (MMP)-1, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 expression were analyzed by real time-PCR. The protein expression of MMP-1 and phosphorylated mitogen-activated protein kinases (MAPKs) were detected by western blotting assay. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) staining.

Results

We found that 20–100 μg/mL of PHT did not affect PDL cells proliferation, whereas 50–100 μg/mL of PHT inhibited cell migration. The 50 or 100 μg/mL of PHT decreased the gene and protein expression of MMP-1, but increased the gene expression of TIMP-1. MMP-2 and TIMP-2 were not affected by 20–100 μg/mL of PHT. Further, 20–50 μg/mL of PHT increased ALP expression, but 100 μg/mL of PHT depressed ALP expression. The extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were activated by PHT. JNK and ERK are involved in PHT-regulated migration. JNK plays an essential role in PHT-induced osteogenic differentiation.

Conclusions

MAPK pathway involved in PHT-regulated migration and osteogenic differentiation in human PDL cells.

中文翻译：

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苯妥英通过 MAPK 通路调节人牙周膜细胞的迁移和成骨分化

介绍

牙周愈合需要足够数量的牙周韧带 (PDL) 细胞来重建受损组织。据报道，苯妥英 (PHT) 可促进伤口愈合和细胞外基质沉积，这表明其在牙周愈合中的应用前景广阔。然而，PHT 对 PDL 细胞行为的影响及其潜在机制仍然未知。

方法

培养和鉴定人PDL细胞。我们的研究中使用了20-100 μ g/mL PHT。通过 EdU 测定法确定 PDL 细胞的增殖。伤口愈合测定用于检测细胞迁移。通过实时PCR分析基质金属蛋白酶(MMP)-1、MMP-2、金属蛋白酶组织抑制剂(TIMP)-1和TIMP-2的表达。通过蛋白质印迹法检测MMP-1和磷酸化丝裂原活化蛋白激酶（MAPKs）的蛋白表达。通过碱性磷酸酶（ALP）染色评估成骨分化。

结果

我们发现 20-100 μg /mL PHT 不影响 PDL 细胞增殖，而 50-100 μg /mL PHT 抑制细胞迁移。50或100μg /mL的PHT降低了MMP-1的基因和蛋白表达，但增加了TIMP-1的基因表达。MMP-2 和 TIMP-2 不受 20-100 μ g/mL PHT 的影响。此外，20-50 μg /mL PHT 增加 ALP 表达，但 100 μg /mL PHT 抑制 ALP 表达。细胞外调节激酶 (ERK)、c-Jun N-末端激酶 (JNK) 和 p38 被 PHT 激活。JNK 和 ERK 参与 PHT 调节的迁移。JNK 在 PHT 诱导的成骨分化中起重要作用。

结论

MAPK通路参与人PDL细胞中PHT调节的迁移和成骨分化。