TLR4通路在软脂酸诱导人血管平滑肌细胞IL-6基因表达中的作用

The role of Toll like receptor 4 pathway in palmitate-induced interleukin-6 expression in human aortic vascular smooth muscle cells

构建Toll样受体4（TLR4）shRNA腺病毒表达载体（pGSadeno—TLR4）并感染人主动脉血管平滑肌细胞（HA-VSMC），采用软脂酸和不同的信号通路阻断剂处理HA—VSMC，以实时定量PCR检测白细胞介素6（IL-6）mRNA的表达，酶联免疫吸附法（ELISA）检测NF—KBp65活性和IL-6的蛋白水平。结果显示，软脂酸增加HA—VSMCIL-6mRNA和蛋白水平，且具有明显的剂量依赖关系，软脂酸（400μmol／L）处理6h作用最明显。IL-6mRNA的表达水平为对照组的10．43倍（P〈0．01）。IL-6蛋白水平在软脂酸作用24h时达到峰值，约为对照组的2．18倍（P〈0．01）。NF．KB阻断剂parthenolide下调软脂酸诱导的IL-6mRNA和蛋白表达分别为65％和59％（均P〈0．01），蛋白激酶C（PKC）阻断剂白屈菜红碱下调软脂酸诱导的IL-6mRNA和蛋白表达分别为24％和28％（均P〈0．05）。而细胞外信号调节激酶抑制剂PD98059和磷酸肌醇3激酶抑制剂wortmannin对软脂酸诱导的IL-6mRNA和蛋白表达均无明显作用（均P〉0．05）。pGSadeno．TLR4下调软脂酸诱导的IL-6mRNA和蛋白表达分别为72％和75％（均P〈0．01），下调软脂酸刺激的NF—KBv65活性约62％（P〈0．01）。这些结果提示TLR-4／NF—KB和PKC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-6基因表达。

The recombinant adenovirus Toll like receptor 4 （ TLR4 ） shRNA vector （ pGSadeno-TLR4 ） was constructed and transfeeted into human aortic vascular smooth muscle cells （ HA-VSMC ）. After HA-VSMC were treated with palmitate or different signaling pathway inhibitors, the mRNA and protein levels of interleukin-6 （ IL-6 ） and NF-KB activity were tested with real-time PCR and ELISA, respectively. The results showed that palmitate increased mRNA and protein levels of IL-6 in HA-VSMC in a dose-dependent manner. The expression of IL-6 mRNA reached peak after treatment with 400μmol/L of palmitate for 6 h, being 10.43 fold of control （ P〈0.01 ）. Treatment with 400 μmol/L of palmitate for 24 h maximally upregulated the protein level of IL-6, which was 2.18 fold of control （ P〈0.01 ）. NF-KB inhibitor parthenolide markedly inhibited palmitate-stimulated increased in IL-6 mRNA level by 65% and protein level by 59% （ both P〈0.01 ）. Protein kinase C （ PKC ） inhibitor chlerythrine suppressed palmitate- induced IL-6 mRNA expression by 24% and IL-6 protein level by 28%. By contrast, extracellular sigual-regulated protein kinase inhibitor PD98059 and phosphatidylinositol 3-kinase inhibitor wortmannin had no effect on the induction of IL-6 by palmitate. Blockade of TLR4 with pGSadeno-TLR4 significantly suppressed palmitate-induced IL-6 mRNA expression by 72% and IL-6 protein expression by 75% （ both P〈0. 01 ）, along with decrease of NF-KB p65 activity decreased by 62%. These results suggest that TLR4/NF-KB and PKC pathways mediate palmitate-induced IL-6 expression in HA-VSMC.