Inhibition effects of nuclear factor-κB on cytokines releasing from human corneal fibroblasts / 解剖学报

ABSTRACT

Objective To investigate the inhibition effects of nuclear factor-κB(NF-κB) on cytokine expression of cultured human corneal fibroblasts in vitro. Methods The cultured human corneal fibroblasts were used in the present experiment. Cell viability insensitive to emodin on cultured human corneal fibroblasts in vitro was assessed using the MTT assay.Cells were randomly divided into three groupscontrol group(group 0 hour,n=6),lipopolysaccharidel (LPS)group (n=24)and emodin pretreatment group(n=24). Cells of the emodin pretreatment group were incubated with emodin for 30 minutes before LPS challenged. The cultured human corneal fibroblasts were then challenged with LPS, the expression of interleukin-8(IL-8) mRNAs was detected by reverse transcription polymerase chain reaction analysis (RT-PCR) and the proteins of IL-8 secreted from these cells were assessed by enzyme-linked immunosorbent assays (ELISA). Western blotting analysis was used for detecting the protein expression of inhibitor of nuclear factor-κB-α(IκB-α)induced by LPS.At the same time,the effects of emodin on these expressions were also assessed in fibroblasts. Results The concentration of emodin used in this study was safe to these cultured human corneal fibroblasts in vitro. Both the protein release and mRNA expression of IL-8 in corneal fibroblasts increased significantly after challenged with LPS, however, the protein level of IκB-α significantly decreased after treated with LPS. These results indicated that LPS could mediate the activation of NF-κB and upregulate the expression of cytokines in corneal fibroblasts. Pretreated with emodin 30 min before challenged with LPS, the activation of NF-κB induced by LPS was markedly inhibited, and the mRNA expression as well as protein release of IL-8 were also inhibited(P<0.01).Conclusion These results suggest that LPS takes part in the degeneration of IκB-α and the expression of IL-8 in cultured human corneal fibroblasts in vitro. Emodin, an active component from the rhizome of Rheum palmatum, can inhibit the activation of NF-κB via decreasing this degeneration, which results in suppressing LPS-induced cytokine release from cultured human corneal fibroblasts.