Effects of amphotericin B on cytokine expressions in epithelial cells

Objectives: Amphotericin B is a polyene anti-fungal agent and has a high affinity for sterols, primary ergosterol of fungal cell membranes.  Amphotericin B has been used for periodontal therapy by some dentists, although the scientific evidence for the effects has not been established.  Periodontitis is inflammatory disease induced by periodontal bacteria.  Since fungus is not involved in periodontal pathogenesis, amphotericin B may regulate host cell function in inflammatory condition.  Gingival epithelium is primary barrier against microorganism invasion, and product inflammatory cytokines such as interleukin (IL)-6 and IL-8.  Thus, the interaction between epithelial cells and bacteria plays an important role in the initial stage of inflammation.  In this study, we investigated the effect of amphotericin B on the expression of inflammatory cytokines in human gingival epithelial cells (HGEC) stimulated by Aggregatibactor actinomycetemcomitans (A.a) or tumor necrosis factor (TNF)-α .

Methods: HGEC were collected from healthy gingival tissue.  Cytotoxic effect of amphotericin B (5 μM, 50 μM, 500μM, 5mM) by MTSassay were analyzed.  HGEC was stimulated by A.a (1×10⁸ cell/ml) or TNF-α (50 ng/ml) in the presence of amphotericin B (50 μM, 500μM).  ERK inhibitor (PD98059) or p38 MAPK inhibitor (SB203580), was added to analyze the signaling pathway.  To investigate anti-inflammatory effects of amphotericin B, IL-6 and IL-8 expression levels were examined by realtimePCR and ELISA, respectively.  The phosphorylation of ERK and p38 MAPK were determined by Western blotting.

Results: Amphotericin B suppressed the expression of IL-6 and IL-8 at the mRNA and protein levels in HGEC by A.a or TNF-α stimulation.  SB203580 suppressed A.a induced up-regulation on IL-6 mRNA, and PD98059 suppressed A.a induced up-regulation on IL-8 mRNA.  In addition, amphotericin B inhibited the phosphorylation of ERK and p38 MAPK induced by A.a. 

Conclusions: These findings suggest that amphotericin B downregulates inflammatory responses of gingival epithelium and may relief clinical symptoms of periodontitis.