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Keywords:

  • human vascular smooth muscle cells;
  • interleukin-8;
  • palmitate;
  • signaling pathway;
  • Toll-like receptor 4
  • 人主动脉血管平滑肌细胞,白细胞介素-8,软脂酸,信号通路,果蝇样受体4

Abstract

Background

Recent evidence demonstrates that saturated free fatty acids (FFAs) induce the inflammatory response via the Toll-like receptor 4 (TLR4) pathway. Interleukin-8 (IL-8) is a proinflammatory cytokine that induces vascular smooth muscle cell proliferation and migration in vitro. However, the regulation of IL-8 expression by palmitate in human vascular smooth muscle cells (HVSMCs) has not been clarified. The aim of this study was to investigate the regulation of IL-8 expression by free fatty acids and determine the underlying mechanisms in HVSMCs.

Methods

Human vascular smooth muscle cells were cultured and treated with palmitate, various signaling inhibitors or TLR4 shRNA adenovirus, and the mRNA and protein expression levels of IL-8, nuclear factor κB (NF-κB) luciferase activity and NF-κB p65 binding activity were studied.

Results

Palmitate induced IL-8 mRNA expression and secretion in a dose-dependent manner. Palmitate significantly stimulated both nuclear factor κB (NF-κB) luciferase activity and NF-κB p65 binding activity, which were markedly diminished by pretreatment with the NF-κB inhibitor, parthenolide. Parthenolide pretreatment also abolished IL-8 mRNA and protein induction by palmitate. By contrast, disrupting the ceramide and phosphoinositide-3 kinase (PI3K) pathways with myriocin and wortmannin did not affect palmitate-induced IL-8 expression. Inhibition of protein kinase C (PKC) activation with calphostin C and chelerythrine partially suppressed palmitate-stimulated IL-8 expression, but it had no effect on palmitate-induced NF-κB activation. Finally, knockdown of TLR4 markedly abolished palmitate-induced NF-κB activation and IL-8 expression.

Conclusions

Palmitate induces IL-8 gene expression in HVSMCs through the TLR4/NF-κB pathway.

摘要

背景

最近的研究表明果蝇样受体4(Toll-like receptor 4,TLR4)通路介导了饱和游离脂肪酸诱导的炎症反应。白细胞介素8(Interleukin-8,IL-8)是一种促炎症因子,能诱导体外培养的血管平滑肌细胞增殖和迁移。但是软脂酸对人血管平滑肌细胞IL-8基因表达的调节作用尚未阐明。本研究旨在探讨游离脂肪酸对人主动脉血管平滑肌细胞IL-8基因表达的调节及其潜在的机制。

方法

采用软脂酸、不同的信号通路阻断剂或TLR4 shRNA腺病毒处理体外培养的人主动脉血管平滑肌细胞,然后检测IL-8 mRNA、IL-8蛋白表达水平、核因子(nuclear factor,NF)-κB荧光素酶活性以及NF-κB p65亚基结合活性。

结果

软脂酸呈剂量依赖性促进IL-8 mRNA的表达和分泌。软脂酸显著激活NF-κB荧光素酶活性和 NF-κB p65结合活性,但采用NF-κB阻断剂parthenolide预处理可显著减弱这种激活。Parthenolide预处理还能阻断软脂酸促进的IL-8 mRNA和蛋白表达。相反,神经酰胺通路阻断剂myriocin和磷酸肌醇-3激酶阻断剂wortmannin对软脂酸诱导的IL-8 表达均无明显作用。抑制蛋白激酶C活性的calphostin C和白屈菜红碱可部分抑制软脂酸诱导的IL-8表达,但对软脂酸诱导的NF-κB激活无明显作用。采用TLR4 shRNA腺病毒沉默TLR4基因表达显著阻断软脂酸诱导 NF-κB激活和IL-8基因表达。

结论

TLR-4/NF-κB信号通路介导了软脂酸诱导的人主动脉血管平滑肌细胞IL-8基因表达。