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Abstract：
OBJECTIVE: Fluocinolone acetonide (FA) is commonly used as a steroidal anti-inflammatory drug. We recently found that in dental pulp cells (DPCs) FA has osteo-/odonto-inductive as well as anti-inflammatory effects. However, the mechanism by which FA induces these effects in DPCs is poorly understood.
METHODS: The effect of FA on the mineralization of DPCs during inflammatory conditions and the underlying mechanism were investigated by real-time PCR, Western blot, EMSA, histochemical staining, immunostaining and pathway blockade assays.
RESULTS: FA significantly inhibited the inflammatory response in LPS-treated DPCs not only by down-regulating the expression of pro-inflammation-related genes, but also by up-regulating the expression of the anti-inflammatory gene PPAR-γ and mineralization-related genes. Moreover, histochemical staining and immunostaining showed that FA could partially restore the expressions of alkaline phosphatase, osteocalcin and dentin sialophosphoprotein (DSPP) and mineralization in LPS-stimulated DPCs. Real-time PCR and Western blot analysis revealed that FA up-regulated DSPP and runt-related transcription factor 2 expression by inhibiting the expression of phosphorylated-NF-κB P65 and activating activator protein-1 (AP-1) (p-c-Jun and Fra-1). These results were further confirmed through EMSA, by detection of NF-κB DNA-binding activity and pathway blockade assays using a NF-κB pathway inhibitor, AP-1 pathway inhibitor and glucocorticoid receptor antagonist.
CONCLUSIONS: Inflammation induced by LPS suppresses the mineralization process in DPCs. FA partially restored this osteo-/odonto-genesis process in LPS-treated DPCs and had an anti-inflammatory effect through inhibition of the NF-κB pathway and activation of the AP-1 pathway. Hence, FA is a potential new treatment for inflammation-associated bone/teeth diseases.
METHODS: The effect of FA on the mineralization of DPCs during inflammatory conditions and the underlying mechanism were investigated by real-time PCR, Western blot, EMSA, histochemical staining, immunostaining and pathway blockade assays.
RESULTS: FA significantly inhibited the inflammatory response in LPS-treated DPCs not only by down-regulating the expression of pro-inflammation-related genes, but also by up-regulating the expression of the anti-inflammatory gene PPAR-γ and mineralization-related genes. Moreover, histochemical staining and immunostaining showed that FA could partially restore the expressions of alkaline phosphatase, osteocalcin and dentin sialophosphoprotein (DSPP) and mineralization in LPS-stimulated DPCs. Real-time PCR and Western blot analysis revealed that FA up-regulated DSPP and runt-related transcription factor 2 expression by inhibiting the expression of phosphorylated-NF-κB P65 and activating activator protein-1 (AP-1) (p-c-Jun and Fra-1). These results were further confirmed through EMSA, by detection of NF-κB DNA-binding activity and pathway blockade assays using a NF-κB pathway inhibitor, AP-1 pathway inhibitor and glucocorticoid receptor antagonist.
CONCLUSIONS: Inflammation induced by LPS suppresses the mineralization process in DPCs. FA partially restored this osteo-/odonto-genesis process in LPS-treated DPCs and had an anti-inflammatory effect through inhibition of the NF-κB pathway and activation of the AP-1 pathway. Hence, FA is a potential new treatment for inflammation-associated bone/teeth diseases.
摘要：
目的：氟轻松（FA）通常用作类固醇类抗炎药。我们最近发现，在牙髓细胞（DPC）中，FA具有骨/齿诱导以及抗炎作用。然而,FA在DPC中诱导这些作用的机制知之甚少。
方法：通过实时PCR研究了FA在炎症状态下对DPCs矿化的影响及其潜在机制，蛋白质印迹,EMSA,组织化学染色,免疫染色和途径阻断测定。
结果：FA不仅通过下调促炎症相关基因的表达来显著抑制LPS处理的DPCs的炎症反应，还可以通过上调抗炎基因PPAR-γ和矿化相关基因的表达。此外，组织化学染色和免疫染色显示，FA可以部分恢复碱性磷酸酶的表达，LPS刺激的DPC中的骨钙蛋白和牙本质唾液酸磷蛋白（DSPP）和矿化。实时PCR和Westernblot分析表明，FA通过抑制磷酸化NF-κBP65和激活激活蛋白1（AP-1）（p-c-Jun和Fra-1）的表达来上调DSPP和runt相关转录因子2的表达。这些结果通过EMSA得到进一步证实，通过使用NF-κB途径抑制剂检测NF-κBDNA结合活性和途径阻断测定，AP-1通路抑制剂和糖皮质激素受体拮抗剂。
结论：LPS诱导的炎症抑制了DPC的矿化过程。FA在LPS处理的DPC中部分恢复了这种骨/牙生过程，并通过抑制NF-κB途径和激活AP-1途径具有抗炎作用。因此，FA是炎症相关骨/牙齿疾病的潜在新疗法。
方法：通过实时PCR研究了FA在炎症状态下对DPCs矿化的影响及其潜在机制，蛋白质印迹,EMSA,组织化学染色,免疫染色和途径阻断测定。
结果：FA不仅通过下调促炎症相关基因的表达来显著抑制LPS处理的DPCs的炎症反应，还可以通过上调抗炎基因PPAR-γ和矿化相关基因的表达。此外，组织化学染色和免疫染色显示，FA可以部分恢复碱性磷酸酶的表达，LPS刺激的DPC中的骨钙蛋白和牙本质唾液酸磷蛋白（DSPP）和矿化。实时PCR和Westernblot分析表明，FA通过抑制磷酸化NF-κBP65和激活激活蛋白1（AP-1）（p-c-Jun和Fra-1）的表达来上调DSPP和runt相关转录因子2的表达。这些结果通过EMSA得到进一步证实，通过使用NF-κB途径抑制剂检测NF-κBDNA结合活性和途径阻断测定，AP-1通路抑制剂和糖皮质激素受体拮抗剂。
结论：LPS诱导的炎症抑制了DPC的矿化过程。FA在LPS处理的DPC中部分恢复了这种骨/牙生过程，并通过抑制NF-κB途径和激活AP-1途径具有抗炎作用。因此，FA是炎症相关骨/牙齿疾病的潜在新疗法。
