Effect Of Interleukin-8Production Induced By Human Neutrophil Peptide-1in HUVEC And The Mechanism Of Signaling Pathway

Objective:To investigate the effect of interleukin-8production induced by human neutrophil peptide-1and related mechanism.Methods:1. Human umbilical vein endothelial cells were stimulated with various human neutrophil peptide-1concentrations (0.625μg/ml,1.250μg/ml,2.500μg/ml,5.000μg/ml,10.10μg/ml)forl2h, and stimulated with10.10μg/ml for different times (Oh、3h、6h、9h、12h), total RNA was extracted, reverse transcription polymerase chain reaction was used to assess relative expression level of interleukin-8mRNA.2. The expression of P2Y6mRNA was indentified by reverse transcription polymerase chain reaction, human umbilical vein endothelial cells were incubated with either the specific inhibitor of P2Y6nucleotide receptor MRS257830min prior to human neutrophil peptide-1stimulation or human neutrophil peptide-1alone, the expression of interleukin-8mRNA was detected by reverse transcription polymerase chain reaction.3. Firstly, human umbilical vein endothelial cells were incubated with either the specific inhibitor of MEK PD98059、specific inhibitor of PI3K LY29400230min prior to human neutrophil peptide-1stimulation or human neutrophil peptide-1alone, the expression of interleukin-8mRNA was detected by reverse transcription polymerase chain reaction. Secondly, western blot was used to detect phosphorylated and total levels of ERK1/2and AKT after human neutrophil peptide-1stimulation.4. Human umbilical vein endothelial cells were incubated with either the specific inhibitor of P2Y6nucleotide receptor MRS257830min prior to human neutrophil peptide-1stimulation or human neutrophil peptide-1alone, phosphorylated and total levels of ERK1/2were detected by western blot.5. Human umbilical vein endothelial cells were incubated with either the specific inhibitor of MEK PD98059、specific inhibitor of PI3K LY29400230min prior to human neutrophil peptide-1stimulation or human neutrophil peptide-1alone, phosphorylated and total levels of ERK1/2and AKT were detect by western blot respectively.Results:1. The interleukin-8expression increased with time lapsing within12h, compared with the negative control group, the interleukin-8expression was significantly higher in each time point(3h、6h、9h、12h)(P<0.05). The interleukin-8expression increased with the increase of human neutrophil peptide-1concentrations, and the interleukin-8expression in5μg/ml and10μg/ml human neutrophil peptide-1treatment groups were significantly higher(P<0.05).2. Human umbilical vein endothelial cells can express P2Y6mRNA stably regardless of human neutrophil peptide-1stimulation. The interleukin-8expression decreased slightly after incubation by the specific inhibitor of P2Y6nucleotide receptor MRS25781μM30min prior to human neutrophil peptide-1stimulation compared with the positive control group (incubated with human neutrophil peptide-1alone)(P>0.05), and in5μM MRS2578treatment group, the interleukin-8expression decreased significantly (P<0.05), although was higher compared with the negative control group(P<0.05).3. The interleukin-8expression induced by human neutrophil peptide-1decreased significantly after incubation by the specific inhibitor of MEK PD98059or specific inhibitor of PI3K LY29400230min prior to human neutrophil peptide-1stimulation compared with the positive control group(P<0.05). The phosphorylation of ERK1/2after human neutrophil peptide-1stimulation enhanced after human neutrophil peptide-1stimulation especially in5min(P<0.05), and weakend with time lapsing. Morever, the phosphorylation of AKT maintained stability.4. The phosphorylation of ERK1/2after human neutrophil peptide-1stimulation for5min enhanced significantly(P<0.05), and weakend slightly when incubated by the specific inhibitor of P2Y6nucleotide receptor MRS25781μM30min prior to human neutrophil peptide-1stimulation (P>0.05), morever, the phosphorylation of ERK1/2weakend significantly in5μM MRS2578treatment group(P<0.05).5. The phosphorylation of ERK1/2rather than AKT enhanced significantly after human neutrophil peptide-1stimulation for5min(P<0.05). The phosphorylation of AKT was almost completely suppressed after incubation by the specific inhibitor of PI3K LY29400230min prior to human neutrophil peptide-1stimulation(P<0.05), and phosphorylation of ERK1/2weakend significantly as well(P<0.05). After incubation by the specific inhibitor of MEK PD9805930min prior to human neutrophil peptide-1stimulation, the phosphorylation of ERK1/2was almost completely suppressed (P<0.05), nevertheless, the phosphorylation of AKT maintained stability (P>0.05).Conclusions:1. Human neutrophil peptide-1can induce interleukin-8production of human umbilical vein endothelial cells time-dependently and dose-dependently within certain time and concentration range.2. Human umbilical vein endothelial cells may induce interleukin-8production through P2Y6nucleotide receptor and PI3K-MAPK/ERK pathways.24charts,13tables,49references.