果蝇样受体4介导游离脂肪酸调节人血管平滑肌细胞单核细胞趋化蛋白-1的表达

Palmitate induces monocyte chemoattractant protein-1 expression in human aortic vascular smooth muscle cells via toll like receptor 4 signaling pathway

目的探讨软脂酸对人主动脉血管平滑肌细胞（HA—VSMC）单核细胞趋化蛋白-1（MCP-1）基因表达的调节及果蝇样受体4（TLR4）信号通路的作用。方法采用不同剂量的软脂酸（100、200、400μmol/L）处理HA—VSMC6、12、24h，采用实时荧光定量聚合酶链反应（PCR）检测细胞MCP．1mRNA表达，酶联免疫吸附实验（ELISA）检测MCP-1蛋白表达，观察软脂酸调节MCP-1表达的剂量依赖关系和时间效应；采用蛋白激酶C（PKC）抑制剂白屈菜红碱、磷脂酰肌醇3激酶（P13K）抑制剂wortmannin、神经酰胺抑制剂myriocin和核因子KB（NF—KB）抑制剂parthenolide分别处理细胞30min，再加入软脂酸（400μmol/L）处理细胞6h后，实时荧光定量PCR检测MCP-1mRNA的表达水平，ELISA检测MCP-1的蛋白表达水平，研究软脂酸调节MCP-1表达依赖的信号通路；构建TLR4短发夹RNA（shRNA）腺病毒pGSadeno-TLR4并感染HA—VSMC沉默TLR4基因表达，实验同时设空白对照组（PBS缓冲液）和对照病毒（pGSadeno-GFP）组。细胞感染96h后更换培养基，再加入软脂酸（400μmoL／L）处理细胞6h，采用实时荧光定量PCR检测MCP-1mRNA表达水平，ELISA检测MCP-1的蛋白表达水平。提取细胞核蛋白，采用ELISA检测NF—KBp65亚基活性，观察TLR4／NF-KB通路在软脂酸调节HA-VSMCMCP-1基因表达中的作用。组间均数比较采用单因素方差分析。结果采用软脂酸处理细胞6h后，对照组及100、200和400μmoVL软脂酸组MCP-1mRNA表达分别为1．00±0．02、3．30±2．84、8．21±4．31和11．25±2．73（F=7．57，P〈0．05）；MCP-1蛋白表达分别为（127±10）、（147±10）、（163±18）和（194±14）ng／L（F=7．81，P〈0．05）。软脂酸可促进HA-VSMCMCP-1mRNA和蛋白表达且具有明显的剂量依赖关系。细胞培养12h和24h后，随着软脂酸浓度的增加，MCP-1mRNA和蛋白表达水平呈逐渐增加趋势，但时间效应则不明显。采用不同的信号通路抑制剂和软脂酸处理细胞6h后，对照组、软脂酸组、软脂酸＋parthenolide组、软脂酸＋白屈菜红碱组、软脂酸＋wortmannin组和软脂酸＋myriocin组MCP-1mRNA表达分别为1．00±0．02、10．80±1．23、3．49±0．28、10．84±0．24、11．24±0．27和10．62±0．36（F=1313．07，P〈0．05）；MCP-1蛋白表达分别为（132±8）、（218±12）、（152±4）、（213±12）、（225±7）和（226±9）ng／L（F=106．83，P〈0．05）。成功地构建并获得TLR4shRNA腺病毒pGSadeno—TLR4，采用pGSadeno-TLR4感染HA—VSMC阻断TLR4信号后，软脂酸＋pGSadeno．TLR4组的NF—KB065结合活性、MCP-1mRNA和蛋白表达均显著低于软脂酸组[分别为0．48±0．12比1．24±0．16、1．88±0．33比10．80±1．23、（154±10）比（218±12）ng／L；F=591．86、659．16、118．37，均P〈0．01]。而对照病毒pGSadeno．GFP对软脂酸诱导的NF—KB065结合活性和MCP：I表达均无明显影响。结论TLR4／NF—KB信号通路介导了软脂酸诱导的人主动脉血管平滑肌细胞MCP-1基因表达。

Objective To investigate the role of toll like receptor 4 （TLR4） pathway in regulation of monocyte ehemoattractant protein-1 （ MCP-1 ） by palmitate in human aortic vascular smooth muscle cells （HA-VSMC）. Methods To study the time course and dose dependent effects of palmitate on MCP-1 gene expression, HA-VSMC were cultured and treated with 100, 200 or 400 p, mol/L of palmitate for 6,12 or 24 h, cells were then harvested at indicated time points, MCP-1 mRNA expression was analyzed with real- time polymerase chain reaction（RT-PCR） and the production of MCP-1 protein in cultural supernatant were tested using enzyme-linked immunosorbent assay （ELISA）. To determine the involvement of signaling pathways in regulation of MCP-1 by palmitate, specific inhibitors that block protein kinase C （PKC）, phosphotylinosital 3 kinase （PI3 K）, ceramide or nuclear factor（NF）-KB signaling were added to the serum- free media for 30 min followed by treatment with or without 400 μmol/L of palmitate for additional 6 h, thereafter, the mRNA and protein expression of MCP-1 were studied. To further clarify the role of TLR4 pathway in induction of MCP-1 by palmitate in HA-VSMC, recombinant adenovirus TLR4 shRNA virus （pGSadeno-TLR4） was constructed, and HA-VSMC were infected with pGSadeno-TLR4 to knockdown the expression of TLR4, infection with pGSadeno-GFP served as a negative control and the cells treated with phosphate buffer saline（PBS） were used as normal control. Ninety-six hours after infection, virus-containing medium was replaced with fresh DMEM medium, the cells were then cultured in the presence or absence of 400 μmol/L of palmitate for 6 h, nuclear protein of the cells was subsequently isolated and NF-KB p65 subunit binding activity was detected with ELISA, MCP-1 mRNA and protein expression were determined. The single-factor analysis of variance was used in data comparison among the groups. Results Six hours after treatment, the mRNA expression of MCP-1 in control group and 100,200,400μmol/L palmitate group were 1.00 ~ 0. 02, 3.30 ± 2. 84, 8.21 ± 4. 31 and 11.25 ± 2. 73, respectively （ F = 7.57, P 〈 0.05 ） ; the protein expression of MCP-1 were （127 ± 10）, （147 ± 10）, （163 ± 18） and （194 ± 14） ng/L, respectively （F = 7.81, P 〈 0. 05 ）. Palmitate significantly induced mRNA and protein expression of MCP-1 in a dose dependent manner, while the time dependent effect was not obvious, these effects were also observed when the cells were treated with palmitate for 12 h or 24 h. Six hours after treatment with various inhibitors of different signaling pathway, the MCP-1 mRNA level in control group, palmitate group, palmitate ± parthenolide group, palmitate ± chelerythrine group, palmitate ± wortmannin group and palmitate ± myriocin group were 1.00 ±0.02, 10.80 ± 1.23, 3.49 ±0.28, 10.84 ±0.24, 11.24 ±0.27 and 10.62±0.36, respectively（F = 1313.07, P 〈 0. 05 ） ; the protein expression of MCP-1 were （ 132 ± 8）, （218 ± 12）, （152±4）, （213 ± 12）, （225 ±7） and （226±9） ng/L, respectively（F= 106. 83, P 〈0.05）. Knockdown of TLR4 with pGSadeno-TLR4 significantly inhibited palmitate induced increase in mRNA and protein expression of MCP-1 and NF-KB p65 binding activity （ 1.88 ±0. 33 vs 10. 80 ± 1.23, （ 154 ± 10） vs （218 ± 12） ng/L, 0. 48 ± 0. 12 vs 1.24 ± 0. 16, F = 591.86, 659. 16, 118.37, all P 〈 0. 05）, while the control virus pGSadeno-GFP implicated no effects. Conclusions Palmitate may induce MCP-1 expression in HA-VSMC through activation of TLR4/NF-KB pathway.