Exendin-4 ameliorates oxidized-LDL-induced inhibition of macrophage migration in vitro via the NF-κB pathway
Abstract
Ge-fei MA#, Song CHEN#, Lei YIN, Xiang-dong GAO*, Wen-bing YAO*
State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
Aim: To investigate the effects of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 on oxidized low-density lipoprotein (ox-LDL)-induced inhibition of macrophage migration and the mechanisms underlying the effects of exendin-4.
Methods: Primary peritoneal macrophages were extracted from the peritoneal cavity of mice treated with 3% thioglycollate (2 mL, ip). Migration of the macrophages was examined using a cell migration assay. Macrophage migration-related factors including leptin-like ox-LDL receptor (LOX-1), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin-1 (IL-1)β, matrix metalloproteinase-2 (MMP-2), intercellular adhesion molecule (ICAM)-1 and macrophage migration inhibitory factor (MIF) were measured using semi-quantitative RT-PCR. Expression of MIF and ICAM-1 proteins was examined with ELISA. Gelatin zymography was used to evaluate the activity of MMP-9. Activation of the NF-κB pathway was determined by confocal laser scanning microscopy.
Results: Treatment of the macrophages with ox-LDL (50 μg/mL) markedly suppressed the macrophage migration. Furthermore, ox-LDL treatment substantially increased the expression of the macrophage migration-related factors, the activity of MMP-9 and the translocation of the NF-κB p65 subunit. These effects of ox-LDL were significantly ameliorated by pretreatment with the specific NF-κB inhibitor ammonium pyrrolidine dithiocarbamate (100 μmol/L). These effects of ox-LDL were also significantly ameliorated by pretreatment with exendin-4 (25 and 50 nmol/L).
Conclusion: Exendin-4 ameliorates the inhibition of ox-LDL on macrophage migration in vitro, via suppressing ox-LDL-induced expression of ICAM-1 and MIF, which is probably mediated by the NF-κB pathway.
Keywords: macrophage; macrophage migration inhibitory factor; ICAM-1; NF-κB; GLP-1; exendin-4; ox-LDL; ammonium pyrrolidine dithiocarbamate; CD36; atherosclerosis
This work was supported by grants from the National Natural Science Foundation of China (81273496 and 81172974), the “Simcere Innovation Fund” (CX11B-004XS), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (SKLNMZZ201211), the “333” Project of Jiangsu Province, and the National High Technology Research and Development Program of China (863 Program) (2013AA092901).
# These two authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail wbyao@cpu.edu.cn (Wen-bing YAO); xdgao@cpu.edu.cn (Xiang-dong GAO)
Received 2013-04-08 Accepted 2013-08-12
Keywords:
State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China
Aim: To investigate the effects of the glucagon-like peptide-1 (GLP-1) receptor agonist exendin-4 on oxidized low-density lipoprotein (ox-LDL)-induced inhibition of macrophage migration and the mechanisms underlying the effects of exendin-4.
Methods: Primary peritoneal macrophages were extracted from the peritoneal cavity of mice treated with 3% thioglycollate (2 mL, ip). Migration of the macrophages was examined using a cell migration assay. Macrophage migration-related factors including leptin-like ox-LDL receptor (LOX-1), cyclooxygenase 2 (COX-2), tumor necrosis factor (TNF)-α, interleukin-1 (IL-1)β, matrix metalloproteinase-2 (MMP-2), intercellular adhesion molecule (ICAM)-1 and macrophage migration inhibitory factor (MIF) were measured using semi-quantitative RT-PCR. Expression of MIF and ICAM-1 proteins was examined with ELISA. Gelatin zymography was used to evaluate the activity of MMP-9. Activation of the NF-κB pathway was determined by confocal laser scanning microscopy.
Results: Treatment of the macrophages with ox-LDL (50 μg/mL) markedly suppressed the macrophage migration. Furthermore, ox-LDL treatment substantially increased the expression of the macrophage migration-related factors, the activity of MMP-9 and the translocation of the NF-κB p65 subunit. These effects of ox-LDL were significantly ameliorated by pretreatment with the specific NF-κB inhibitor ammonium pyrrolidine dithiocarbamate (100 μmol/L). These effects of ox-LDL were also significantly ameliorated by pretreatment with exendin-4 (25 and 50 nmol/L).
Conclusion: Exendin-4 ameliorates the inhibition of ox-LDL on macrophage migration in vitro, via suppressing ox-LDL-induced expression of ICAM-1 and MIF, which is probably mediated by the NF-κB pathway.
Keywords: macrophage; macrophage migration inhibitory factor; ICAM-1; NF-κB; GLP-1; exendin-4; ox-LDL; ammonium pyrrolidine dithiocarbamate; CD36; atherosclerosis
This work was supported by grants from the National Natural Science Foundation of China (81273496 and 81172974), the “Simcere Innovation Fund” (CX11B-004XS), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (SKLNMZZ201211), the “333” Project of Jiangsu Province, and the National High Technology Research and Development Program of China (863 Program) (2013AA092901).
# These two authors contributed equally to this work.
* To whom correspondence should be addressed.
E-mail wbyao@cpu.edu.cn (Wen-bing YAO); xdgao@cpu.edu.cn (Xiang-dong GAO)
Received 2013-04-08 Accepted 2013-08-12