Yan Hui, Zhu Wei-Guo, Li Shan, Fan Fang-Yan, Sun Pei-Yu, Zhu Jian-Hua
Department of Cardiology, First Affiliated Hospital of Zhejiang University, School of Medicine, Hangzhou 310003, China.
Zhonghua Yi Xue Za Zhi. 2008 Mar 25;88(12):835-9.
To investigate the effect of pioglitazone (Pio) on dendritic cell-(DC) specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) expression in DCs and explore the possible mechanism of Pio inhibiting DC adhesion and transmigration.
DCs derived from human peripheral blood mononuclear cells were cultured and divided into 6 groups: blank control group, Pio 0.1 micromol/L group, Pio 1.0 micromol/L group, Pio 10 micromol/L group, GW9662, a peroxisome proliferator activated receptor (PPAR)-gamma antagonist, 10 micromol/L group, and GW9662 10 micromol/L + Pio 10 micromol/L group. Western blotting was used to detect the protein expression of DC-SIGN 24 h later. Human umbilical vein endothelial cells (HUVECs) were obtained and co-cultured with the DCs undergoing different treatments. Immunofluorescence test was used to detect the protein expression of DC-SIGN. DCs labeled with 5-chloromethylfluorescein diacetate (CMFDA) were added into the monocellular layer of fused ECs. Blank DCs and DCs pretreated with anti-DC-SIGN antibody were used as blank and experimental groups. Laser confocal microscopy was used to observe the adhesion ability of the DCs. HUVECs were inoculated into the upper chamber of Transwell plate and CMFDA-labeled DCs of above mentioned groups were added to the mono-cellular layer of these ECs. Serum-free culture medium with monocyte chemoattractant protein-1 was added into the lower chamber. Eight hours later the transmigration ability was observed.
Western blotting showed that the DC-SIGN protein expression levels of the DCs of the Pio 1.0 micromol/L and Pio 10 micromol/L group were 0.96 +/- 0.09 and 0.80 +/- 0.08 respectively, both significantly lower than that of the blank control group (1.25 +/- 0.23, P < 0.05 and P < 0.01); and the DC-SIGN protein expression level of the GW9662 10 micromol/L + Pio 10 micromol/L group was 1.10 +/- 0.12, significantly higher than that of the Pio 10 micromol/L group (P < 0.05). Immunofluorescence test showed that the DC-SIGN protein expression levels of the DCs of the Pio 1.0 micromol/L and Pio 10 micromol/L group were 22.3 +/- 5.4 and 14.4 +/- 2.3 respectively, both significantly lower than that of the control group (29.5 +/- 5.1, P < 0.05 and P < 0.01), and the DC-SIGN protein expression level of the GW9662 10 micromol/L + Pio 10 micromol/L group was 24.9 +/- 4.3, significantly higher than that of the Pio 10 micromol/L group (P < 0.01), and not significantly different from that of the blank control group (P > 0.05). The adhesion rates of the Pio 1.0 micromol/L and Pio 10 micromol/L groups were 10.8% +/- 2.0% and 7.6% +/- 1.5% respectively, both significantly lower than that of the control group (13.4% +/- 2.1%, P < 0.05 and P < 0.01); and the adhesion rate of the GW9662 + Pio 10 micromol/L group was 12.1% +/- 1.9%, significantly higher than that of the Pio 10 micromol/L group (P < 0.01), and not significantly different from that of the blank control group (P > 0.05). The transmigration inhibition rate of DCs of the Pio 0.1, 1.0, and 10 micromol/L groups were 4.1%, 12.9%, and 17.2% compared with the transmigration rate of the blank control group (P < 0.05, P < 0.05, and P < 0.01). The transmigration rate of the GW9662 + Pio 10 micromol/L group was significantly higher than that of the Pio 10 micromol/L group (P < 0.05), and not significantly different from that of the blank control group (P > 0.05). The transmigration rate of the anti-DC-SIGN intervention group was lower by 17.8% than that of the control group (P < 0.01).
Pio down-regulates the DC-SIGN protein expression and inhibits DC adhesion and transmigration through the pathway of PPAR-gamma.
探讨吡格列酮（Pio）对树突状细胞（DC）特异性细胞间黏附分子3结合非整合素分子（DC-SIGN）表达的影响，探讨Pio抑制DC黏附和迁移的可能机制。
培养来源于人外周血单个核细胞的DC，并分为6组：空白对照组、0.1 μmol/L Pio组、1.0 μmol/L Pio组、10 μmol/L Pio组、过氧化物酶体增殖物激活受体（PPAR）-γ拮抗剂GW9662 10 μmol/L组、GW9662 10 μmol/L + Pio 10 μmol/L组。24小时后采用蛋白质印迹法检测DC-SIGN的蛋白表达。获取人脐静脉内皮细胞（HUVEC），并与经过不同处理的DC共培养。采用免疫荧光试验检测DC-SIGN的蛋白表达。将用5-氯甲基荧光素二乙酸酯（CMFDA）标记的DC加入融合内皮细胞的单细胞层中。空白DC和用抗DC-SIGN抗体预处理的DC分别作为空白组和实验组。采用激光共聚焦显微镜观察DC的黏附能力。将HUVEC接种于Transwell小室上室，并将上述各组CMFDA标记的DC加入这些内皮细胞的单细胞层中。在下室加入含单核细胞趋化蛋白-1的无血清培养基。8小时后观察迁移能力。
蛋白质印迹法显示，1.0 μmol/L Pio组和10 μmol/L Pio组DC的DC-SIGN蛋白表达水平分别为0.96±0.09和0.80±0.08，均显著低于空白对照组（1.25±0.23，P<0.05和P<0.01）；10 μmol/L GW9662 + 10 μmol/L Pio组的DC-SIGN蛋白表达水平为1.10±0.12，显著高于10 μmol/L Pio组（P<0.05）。免疫荧光试验显示，1.0 μmol/L Pio组和10 μmol/L Pio组DC的DC-SIGN蛋白表达水平分别为22.3±5.4和14.4±2.3，均显著低于对照组（29.5±5.1，P<0.05和P<0.01），10 μmol/L GW9662 + 10 μmol/L Pio组的DC-SIGN蛋白表达水平为24.9±4.3，显著高于10 μmol/L Pio组（P<0.01），与空白对照组无显著差异（P>0.05）。1.0 μmol/L Pio组和10 μmol/L Pio组的黏附率分别为10.8%±2.0%和7.6%±1.5%，均显著低于对照组（13.4%±2.1%，P<0.05和P<0.01）；10 μmol/L GW9662 + 10 μmol/L Pio组的黏附率为12.1%±1.9%，显著高于10 μmol/L Pio组（P<0.01），与空白对照组无显著差异（P>0.05）。与空白对照组的迁移率相比，0.1、1.0和10 μmol/L Pio组DC的迁移抑制率分别为4.1%、12.9%和17.2%（P<0.05、P<0.05和P<0.01）。10 μmol/L GW9662 + 10 μmol/L Pio组的迁移率显著高于10 μmol/L Pio组（P<0.05），与空白对照组无显著差异（P>0.05）。抗DC-SIGN干预组的迁移率比对照组低17.8%（P<0.01）。
Pio通过PPAR-γ途径下调DC-SIGN蛋白表达，抑制DC黏附和迁移。
