Effect Of Platelet-activating Factor On Src-suppressed C Kinase Substrate Gene Expression In Rat Pulmonary Microvascular Endothelial Cells

Objective: To investigate the effect of platelet-activating factor(PAF) on the production of Src-suppressed C kinase substrate(SSeCKS) mRNA in rat pulmonary microvascular endothelial cells(RPMVEC) and to observe the effect of signal inhibitors on inflammation-induced SSeCKS gene expression. On the basis of our previous work, to explore the mechanism of PAF involved in ALI and initially study the signal transduction pathways of SSeCKS gene expression in the inflammation-induced RPMVEC injury process.Methods: RPMVEC were isolated and cultured in vitro, then according to the different culture dosage or time of PAF randomly grouped into two sections. One was using 10-10, 10-9, 10-8, 10-7 mol/L PAF incubated cells for 1.5 h, the other was using 10-7 mol/L PAF challenge for 0.5, 1.5, 3, 6, 12, 24 h. Pretreatment of 10μmol/L nuclear factor-kappa B(NF-κB) inhibitor pyrrolidine dithiocarbamate(PDTC) or protein kinase C(PKC) inhibitor bis-indolylmaleimide(BIM) was used to interfere PAF and LPS stimulation. The normal, negative and positive control groups were set concurrently. In situ hybridization(ISH) and computer image analysis software was jointly performed to reveal the change of SSeCKS mRNA expression in the cultured RPMVECs under each condition.Results: 1. We have isolated and cultured the primary RPMVEC in vitro.These cells were further confirmed by typical morphology and FITC-BSI binding assay. 2. We have established a method for the detection of SSeCKS mRNA in RPMVEC by ISH approach using digoxin-labelled oligonucleotide probes. 3. Normal RPMVEC expressed SSeCKS mRNA at a low level, which appeared throughout the cytoplasm with specific hybridization signals. 1.5 h of 10-10, 10-9, 10-8, 10-7 mol/L PAF incubation induced a progressive increase in SSeCKS mRNA expression. When compared SSeCKS mRNA's mean optical density with the normal control group, difference in each group had statistically significance. Within 0.5, 1.5, 3, 6, 12, 24 h of 10-7 mol/L PAF challenge, the level of SSeCKS mRNA expression markedly raised at 0.5 h, peaked at 1.5 h, then began to decline gradually, and still persisted at a higher level than the normal control group until 24 h. 4. Pre-incubation of 10μmol/L pyrrolidine dithiocarbamate(PDTC) that inhibits activity of nuclear factor-kappa B(NF-κB) in RPMVECs caused a conspicuous attenuation of PAF- or LPS-induced SSeCKS mRNA expression, whereas no change was found by pretreatment of protein kinase C(PKC) inhibitor bis-indolylmaleimide(BIM).Conclusion: 1. We have successfully cultured and demonstrated the primary RPMVEC in vitro. 2. Through the combination of ISH and computer image analysis software ,We have successfully detected the change of SSeCKS gene expression under different conditions in RPMVEC. 3. PAF can up regulate expression of SSeCKS mRNA in a dose- and time-dependent manner in RPMVECs. 4. It is NF-κB rather than PKC signal pathway that involve in modulation of the intracellular signaling process that induce SSeCKS mRNA expression.