Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction
Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction
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DOI：
10.1111/odi.12651
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发表时间：
2017-07-01
期刊：
影响因子：
3.8
通讯作者：
Hiratsuka, K.
中科院分区：
文献类型：
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作者：
Takeuchi, R.;Matsumoto, H.;Hiratsuka, K.
目的探讨苯妥英钠(phytoin，PHT)在死亡受体诱导的牙龈成纤维细胞凋亡中的作用，以阐明PHT诱导牙周成纤维细胞过度生长的机制。方法将人牙龈成纤维细胞培养至半融合状态，加入苯妥英钠(0.025.5、0.1、0.2 5、1.0M)处理48h后，用吸光度法检测细胞凋亡数。0.25M PHT作用24小时后，用吸光度法检测caspase活性，流式细胞仪检测细胞周期和细胞周期时相分布，实时荧光定量聚合酶链式反应检测细胞凋亡基因表达水平，Western印迹法检测细胞凋亡蛋白表达。结果与无血清对照组相比，0.25M PHT作用48h后，牙龈成纤维细胞的凋亡细胞比例降低，其作用机制如下：PHT上调c-Flip，进而下调FADD、caspase-8和caspase-3；PHT上调c-IAP2，下调TRAF2；PHT通过降低RIPK1活性和增加Bc-2活性，下调caspase-9和caspase-3的表达。结论PHT诱导牙周成纤维细胞过度生长的机制可能与其抑制牙龈成纤维细胞的凋亡有关。
ObjectiveIn this study, we investigated the role of phenytoin (PHT) in death receptor-induced apoptosis of gingival fibroblasts to clarify the mechanism of PHT-induced gingival overgrowth.MethodsHuman gingival fibroblasts were cultured to semiconfluence and treated with PHT (0.025, 0.1, 0.25, and 1.0M) for 48h, and then, the apoptotic cell numbers were relatively determined by absorptiometry. After 24h of 0.25M PHT treatment, caspase activity was measured by absorptiometry, apoptotic and cell cycle phase distribution was analyzed by flow cytometry, expression levels of apoptotic genes were quantified by real-time qPCR, and expression of apoptotic proteins was detected by Western blot analysis. After 48h of 0.25M PHT treatment, appearance of apoptotic cells was detected by TUNEL assay.ResultsPHT treatment decreased the proportion of apoptotic cells in gingival fibroblasts compared to a serum-free control culture in response to the protein changes as follows: PHT upregulated c-FLIP and, in turn, downregulated FADD, caspase-8, and caspase-3; PHT upregulated c-IAP2 and downregulated TRAF2; PHT downregulated caspase-9 and caspase-3 via decreased RIPK1 activity and increased Bcl-2 activity.ConclusionPHT-induced gingival overgrowth may result from the above-mentioned mechanisms involving apoptosis inhibition in gingival fibroblasts.