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Objective To explore the possible role of neuropilin-1 (NRP-1) and extracellular regulated protein kinases (ERK) in the mechanisms underlying the promotive effect of vascular endothelial growth factor (VEGF) on the proliferation of HaCaT cells.Methods HaCaT cells were cultured in vitro and transfected with a NRP-1 expression plasmid EX-O0008-M02.Reverse transcription (RT)-PCR and Western blot were performed to detect the mRNA and protein expression of NRP-1 in HaCaT cells respectively before and after the transfection.Some HaCaT cells were divided into three groups to be transfected with liposome (liposome control group),control plasmid pReceiver-M02 (plasmid control group),and objective plasmid EX-O0008-M02 (objective plasmid group),respectively,and each of the three groups was classified into several subgroups to be treated with phosphate buffer solution (PBS),U0126 (MEK1/2 inhibitor) and VEGF alone or in combination.After additional culture,methyl thiazolyl tetrazolium (MTT) assay was performed to determine the proliferative activity of HaCaT cells,Western blot to quantify the expression of total and phosphorylated ERK1/2 as well as proliferating cell nuclear antigen (PCNA) protein in HaCaT cells.Intergroup differences were assessed by one-way analysis of variance (ANOVA),and multiple comparisons and correction were done by using the least significant difference (LSD) test.Results RT-PCR and Western blot analysis confirmed that the transfection with NRP-1 effectively promoted the mRNA and protein expression of NRP-1 in HaCaT cells.A significant increase was observed in cellular proliferative activity (absorbence value at 570 nm) in the objective plasmid group compared with the liposome control group and plasmid control group (0.88 ± 0.14 vs.0.63 ± 0.07 and 0.62 ± 0.13,F =8.755,P ＜ 0.05),also in the VEGF-stimulated objective plasmid group compared with the VEGF-stimulated plasmid control group (1.14 ± 0.18 vs.0.88 ± 0.10,F =4.591,P ＜ 0.05).The U0126 pretreatment markedly suppressed the VEGF-induced proliferation of A375 cells in the objective plasmid group (0.50 ± 0.13 vs.1.14 ± 0.18,F =42.106,P ＜ 0.01).As Western blot analysis suggested,the objective plasmid significantly enhanced the VEGF-induced increase in ERK1/2 phosphorylation degree and PCNA expression intensity in HaCaT cells compared with the control plasmid,but the enhancing effect of objective plasmid was effectively inhibited by U0126.Conclusion The activation of ERK1/2 signaling pathway may play a key role in the NRP-1 protein-mediated promotive effect of VEGF on the proliferation of HaCaT cells.
RESUMO
<p><b>OBJECTIVE</b>To explore the chaotic dynamic process of multiple organs dysfunction syndrome (MODS) and the regulatory effect of Shenqin Liquid (SQL), a Chinese herbal liquid preparation with the action of purging and qi-tonifying.</p><p><b>METHODS</b>Eighty SD rats were divided into 4 groups, and were given suspension of zymosan A and paraffine (1 mL/kg) by peritoneal injection except for those in the blank control group to set up the multiple organs dysfunction syndrome (MODS) model. Low and high doses SQL were administered twice at the doses of 30 and 60 g/kg of SQL respectively at an interval of 8 h per day before modeling. Serum concentration of tumor necrosis factor alpha (TNF-alpha) and nitric oxide (NO) in MODS model animals were tested diachronically, eg. 12, 6 h before modeling, during modeling, 6 and 12 h after modeling, and then the mathematic models were built up with compartment analysis. Lyapunov exponents (LE) of the mathematic models were calculated to evaluate their chaotic characteristics of movement and the degree of chaos was ascertained with the correlation dimension (CD).</p><p><b>RESULTS</b>The serum levels of TNF-alpha and NO were significantly higher than those in the bland control group at modeling, 6, and 12 h after modeling (P <0.01), while those in the low and high doses of SQL were significantly lower than the model group (P <0.01). Moreover, the level of NO in the high dose of SQL was significantly lower than that in the low dose group (P <0. 01). CD of TNF-alpha movement in the blank control group was 0.803 with the LE less than zero; those in the model group was 1. 966 and > 0 respectively; in the low dose and high dose SQL treated groups, CD was 0.517 and 0.653 respectively and LE >0. CD of NO movement in the blank control group was 0.670 and with LE < 0; in the model group, 1.242 with LE > 0; in the low dose SQL group, 0.574 and in the high dose SQL group 0.850, and LE <0 in the two groups.</p><p><b>CONCLUSION</b>Under the normal physiologic condition, TNF-alpha and NO moved steadily without chaotic properties; while under the pathologic condition of MODS, they manifest relatively complicated chaotic properties. SQL can intervene the movement of TNF-alpha and NO, decrease the complexity of their chaotic movement, and make them return back to a stable state.</p>