O'Sullivan Adrian W, Wang Jiang H, Redmond Henry P
Department of Academic Surgery, National University of Ireland and University College Hospital, Cork, Ireland.
J Surg Res. 2009 Jan;151(1):138-44. doi: 10.1016/j.jss.2008.02.031. Epub 2008 Mar 18.
P38 mitogen activated protein kinase (p38 MAPK) is a critical mediator of the inflammatory response, which makes it a suitable candidate as a novel therapeutic strategy for inflammatory conditions. In this study, we set out to examine the precise role of both protein kinase C (PKC) and P38 MAPK signaling kinases in bacterial lipoprotein (BLP) induced nuclear factor-kappa B (NFkappaB) activation and tumor necrosis factor-alpha (TNFalpha) release in THP-1 monocytic cell line.
THP-1 cells were incubated with BLP(0-1000 ng/mL), phorbol myristate acetate (PMA; 0-100 microg/mL) or a combination of both for 6 and 24 h, with or without pretreatment with SB202190, a specific inhibitor of p38 MAPK and bisindolylmaleimide I, a specific inhibitor of PKC (0-200 microm). Cell supernatants were analyzed for TNF-alpha release and apoptosis. NFkappaB activity was analyzed by electromobility supershift assay.
BLP induced TNF-alpha release was significantly reduced by pretreatment with SB202190 at all concentrations (428.7 +/- 5.9 versus 51 +/- 0.8 rhog/mL, P < 0.05). Pretreatment with bis I significantly inhibited TNF-alpha release at higher concentrations (200 microM) (429.7 +/- 5.9 versus 194.9 +/- 42.68 rhog/mL, P < 0.05) but this was much less effective than SB202190. PMA induced TNF-alpha release was not inhibited at 6 h by either SB202190 or bis I, but was significantly so at 24 h (148.5 +/- 9.8 versus 24 +/- 1.7 and 25.1 +/- 4.4 rhog/mL, P < 0.05). BLP or lipopolysaccharide (LPS) did not result in apoptosis in THP-1 cells (P > 0.05) with PMA inducing apoptosis in a time- and dose-dependent manner. In combination with BLP (1000 ng/mL) but not LPS (1000 ng/mL), low dose PMA resulted in a significant increase in apoptosis, 6% +/- 0.5% (Control) versus 9.2% +/- 0.3% (P < 0.05) and 7% +/- 2.2% (Control) versus 7.7% +/- 0.3% (P > 0.05), respectively. This synergistic effect was inhibited by bisindolylmaleimide 100 nm, 8.9% +/- 0.9% (Control) versus 9.8% +/- 0.2% (P > 0.05). PMA and BLP induced rapid nuclear translocation of NFkappaB, which was inhibited by pretreatment with both SB-202190 and bis I, and SB202190 but not bis I, respectively.
P38 is a critical mediator of BLP induced TNF-alpha release and NFkappaB activation, whereas PKC is only partially responsible for its response. P38 and PKC are both critical mediators of PMA induced TNF-alpha release and NFkappaB activation.
p38丝裂原活化蛋白激酶（p38 MAPK）是炎症反应的关键介质，这使其成为炎症性疾病新型治疗策略的合适候选者。在本研究中，我们着手研究蛋白激酶C（PKC）和P38 MAPK信号激酶在细菌脂蛋白（BLP）诱导的THP-1单核细胞系中核因子-κB（NFκB）激活及肿瘤坏死因子-α（TNFα）释放中的精确作用。
将THP-1细胞与BLP（0 - 1000 ng/mL）、佛波酯（PMA；0 - 100 μg/mL）或两者的组合孵育6小时和24小时，有无用SB202190（p38 MAPK的特异性抑制剂）和双吲哚马来酰胺I（PKC的特异性抑制剂，0 - 200 μM）预处理。分析细胞上清液中的TNF-α释放和凋亡情况。通过电泳迁移率超迁移分析检测NFκB活性。
用SB202190预处理在所有浓度下均显著降低BLP诱导的TNF-α释放（428.7±5.9对51±0.8 μg/mL，P < 0.05）。用双吲哚马来酰胺I预处理在较高浓度（200 μM）时显著抑制TNF-α释放（429.7±5.9对194.9±42.68 μg/mL，P < 0.05），但效果远不如SB202190。PMA诱导的TNF-α释放在6小时时未被SB202190或双吲哚马来酰胺I抑制，但在24小时时显著被抑制（148.5±9.8对24±1.7和25.1±4.4 μg/mL，P < 0.05）。BLP或脂多糖（LPS）未导致THP-1细胞凋亡（P > 0.05），而PMA以时间和剂量依赖方式诱导凋亡。与BLP（1000 ng/mL）而非LPS（1000 ng/mL）联合时，低剂量PMA导致凋亡显著增加，分别为6%±0.5%（对照）对9.2%±0.3%（P < 0.05）和7%±2.2%（对照）对7.7%±0.3%（P > 0.05）。这种协同效应被100 nM双吲哚马来酰胺抑制，8.9%±0.9%（对照）对9.8%±0.2%（P > 0.05）。PMA和BLP诱导NFκB快速核转位，分别被SB - 202190和双吲哚马来酰胺I预处理、SB202190但非双吲哚马来酰胺I预处理抑制。
P38是BLP诱导的TNF-α释放和NFκB激活的关键介质，而PKC仅部分参与其反应。P38和PKC都是PMA诱导的TNF-α释放和NFκB激活的关键介质。
