Effect And Mechanism Of Phenformin On The Inflammatory Response In Human Oral Keratinocytes

Objectives:To evaluate the effect of phenformin on the inflammatory response in human oral keratinocytes(HOKs)induced by 12-O-tetradecanoylphorbol-13-acetate(TPA)or Poly(I:C)and explore its mechanism.Methods:(1)In Vitro:human oral keratinocytes were stimulated with 5ug/ml TPA or 10ug/ml Poly(I:C)for 2 hours and then treated with different concentration(0.5mM/L,1.0mM/L,1.5mM/L)phenformin for 22 hours,using Quantitative Real-time polymerase chain reaction(RT-PCR)and enzyme linked immunosorbent assay(Elisa)to detect the expression on RNA level of proinflammatory cytokines including interleukin 6(IL-6),interleukin 8(IL-8),tumor necrosis factor alpha(TNF-a)and activating transcription factor 3(ATF-3);using Western Blot to detect the expression on protein level of ATF-3,adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)and extracellular regulated protein kinases(Erk)in mitogen-activated protein kinase(MAPK)signal pathway;(2)Ex Vivo:human gingival tissue were stimulated with 100ug/ml Poly(I:C)for 2 hours and then treated with different concentration phenformin(1.5mM/L,3.0mM/L,6.0mM/L)for 22 hours,using RT-PCR to detect the expression on RNA level of inflammatory cytokines including IL-6,IL-8 and TNF-a.Results:(1)In Vitro:RT-PCR and Elisa results showed that 5ug/ml TPA,10ug/ml Poly(I:C)and different concentration(0.5mM/L,1.0mM/L,1.5mM/L)phenformin exhibited no toxicity to HOKs,and phenformin inhibited TPA or Poly(I:C)-induced IL-6,IL-8 and TNF-? production in a dose-dependent manner.RT-PCR and Western-Blot results showed that phenformin and Poly(I:C)could synergistically facilitate ATF-3 expression,and phenformin could suppress the expression of Erk in MAPK signal pathway.(2)Ex Vivo:RT-PCR results showed that phenformin inhibited 100ug/ml Poly(I:C)-induced IL-6,IL-8 and TNF-? production in a dose-dependent manner.Conclusion:Phenformin inhibited TPA or Poly(I:C)-induced production of IL-6,IL-8 and TNF-a of the HOSs in a concentration-dependent manner by induction of ATF-3 and suppression of Erk in MAPK signal pathway.Phenformin has a potential clinical application for treating inflammatory diseases such as periodontitis and some other in oral and maxillofacial areas,it may be a novel idea for treatment of inflammatory diseases.