[The effects of the inhibitor of nuclear factor on lipopolysaccharide-induced cytokine expression in cultured human corneal fibroblasts].
OBJECTIVE
To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on Lipopolysaccharide (LPS)-mediated activation of nuclear factor kappa B (NF-kappaB) and cytokine expression in cultured human corneal fibroblasts.


METHODS
It was a experimental study. A completely random design was employed in this research. Human corneal fibroblasts (HCFs) were obtained from human specimen. HCFs were divided into three groups: control group (group 0 h), LPS alone group and PDTC treatment group. Cells were incubated with PDTC for 30 min in the PDTC pretreatment group before LPS challenged. At different time after LPS challenged, the activities of NF-kappaB were assessed by Western Blot analysis, the secretion of IL-6 and IL-8 from cultured corneal fibroblasts was measured with enzyme-linked immunosorbent assays (ELISA); the mRNAs expression of IL-6 and IL-8 was determined by reverse transcription polymerase chain reaction (RT-PCR). The effects of PDTC on activation of NF-kappaB and the expression of IL-6 and IL-8 were also assessed in HCFs challenged with LPS.


RESULTS
Compared with control group, NF-kappaB level was significantly enhanced in the nucleus in the LPS alone group, indicating that LPS mediated the activation of NF-kappaB in HCFs. The activation of NF-kappaB by LPS was markedly inhibited by PDTC (t1h = 9.3766, t2h = 15.9011, t4h = 12.5851, t8h = 10.8346, P < 0.01). Compared with control group, LPS increased IL-6 and IL-8 expression both mRNA and protein in HCFs. At the same time, PDTC partly inhibited the expression of IL-6 and IL-8 in corneal fibroblasts induced by LPS. These inhibitory effects were significant at both the mRNA and protein levels (protein of IL-6: t1h = 7.9154, t2h = 10.863, t4h = 8.2451, t8h = 13.5063. protein of IL-8: t1h = 8.5663, t2h = 20.5169, t4h = 25.1580, t8h = 34.8699. mRNA of IL-6: t1h = 12.0235, t2h = 13.2894, t4h = 24.0799, t8h = 27.2261. mRNA of IL-8: t1h = 20.9424, t2h = 24.1314, t4h = 29.8580, t8h = 47.9442. P < 0.01).


CONCLUSIONS
These results suggest that LPS can activation of NF-kappaB in cultured HCFs and PDTC partly inhibits NF-kappaB activation.
Department of Ophthalmology, Second Hospital, Shandong University, Jinan 25003, China.
Guo-ling
GL
Chen
Xin-yi
XY
Wu
D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011759:Pyrrolidines / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D013859:Thiocarbamates / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D015850:Interleukin-6; D016209:Interleukin-8; D008070:Lipopolysaccharides; D016328:NF-kappa B; D011759:Pyrrolidines; D012333:RNA, Messenger; D013859:Thiocarbamates; D014409:Tumor Necrosis Factor-alpha; C020972:pyrrolidine dithiocarbamic acid