The Influence Of Macrophage Migration Inhibitory Factor On Mouse Adventitial Fibroblasts In Proliferation, Migration And Phenotype Conversion And The Inhibition Of Triptolide

1 Background and purpose:Percutaneous coronary intervention (PCI) has been widely used in the treatment of coronary heart disease, but the restenosis after surgery restricts its application.Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine. Previous studies found, MIF may affect the proliferation and migration of many types of cells, but also can cause the phenotype conversions of MRC-5 cell and NIH 3T3 cell.Tripterygium is a traditional Chinese medicine, the effective monomer composition is triptolide (TP). According to the studies, TP has immune suppression, anti-inflammatory and anti-tumor effect. Recent studies have shown that, TP also promotes apoptosis of a variety of cells.Previous view considered that the pathogenesis of restenosis after vascular injury was vascular smooth muscle cells migrating to the intima, that caused intimal thickening and luminal stenosis. But recent studies found that the proliferation, migration and phenotypic conversion of adventitial fibroblasts, then the synthesis and secretion of extracellular matrix (ECM) ,those result in the formation of scar damage adventitia, recoil and membrane remodeling, that is the important pathogenesis of restenosis after PCI. Therefore, We culture mouse adventitial fibroblasts (MAFB) in vitro, study the roles of MIF and TP in the proliferation, migration and phenotype conversion of MAFB, and dissuss the possible mechanism of TP and MIF in the restenosis.2 Methods:2.1 Culture and identification of MAFBMAFB was cultured by the digestion with collagenaseⅠ,the identification used immunohistochemistry of vimentin.2.2 Proliferation of MAFB after treatment with MIF (CCK-8 method)The proliferation of MAFB after treatment with MIF was detected by CCK-8 method, the growth situation detection of MAFB was arranged on 0,6,12,24,48,60,and 72 hours.2.3 Migration of MAFB after treatment with MIF (transwell dish method) using transwell culture dishes,detected the migration of MAFB after treatment withMIF,and counted the numbers of the migrated cells under a microscope.2.4 Phenotype conversion of MAFB after treatment with MIFThe phenotype conversion assay have three indicators, namely,α-smooth muscle actin (α-SMA), vimentin, and the hydroxyproline of cell culture supernatant. The first two were detected by western blot, the last one was detected by the digestion method of hydroxyproline, and then converted to collagen content.2.5 Proliferation of MAFB after treatment with TPThe proliferation of MAFB was detected by CCK-8 method after treatment with TP, the growth situation detection of MAFB was arranged on 0,6,12,24,48,60,and 72 hours.2.6 Apoptosis of MAFB after treatment with TP:After treatment with TP,stained by Annexin V-FITC and Propidium Iodide, the apoptosis of MAFB was detected by the flow cytometry.2.7 Proliferation, migration and phenotype conversion of MAFB after treatment with MIF and TP:Methods were such as the formers,the experiments set 6 groups:MIF50μg / L group (a), MIF50μg / L + TP12 .5μg / L group (b), MIF50μg / L + TP25μg / L group (c), TP12.5μg / L group (d), TP25μg / L group (e) and control groups (f).2 Results:3.1 MAFB can be successfully cultured by the digestion with collagenaseⅠ. 3.2 MIF (0-100μg / L) promoted the proliferation, migration, collagen synthesis of MAFB in dose-dependent manner.3.3 MIF (0-50μg / L) promoted the phenotype conversion of MAFB, but the influence of MIF has reached a plateau when it's concentration was 50μg / L. The phenotype conversion of MIF was weaker when it's concentration was 100ug / L than 50ug / L, but there was no significance (P> 0.05).3.4 TP (0-50μg / L) took no significant effect on proliferation of MAFB ,TP(100μg / L) could inhibit the proliferation of MAFB.3.5 TP (0-25μg / L) took no significant effect on apoptosis of MAFB, TP(50μg / L) can cause apoptosis of MAFB.3.6 Treated MAFB with the combination of TP (0-25μg / L) and MIF50μg / L,we found that TP could inhibit the proliferation, migration and phenotype conversion induces by MIF in dose-dependent manner.3.7 TP (0-25μg / L) alone took no significant effect on the proliferation, migration and phenotype conversion of MAFB.4 Conclusion:MIF promoted the proliferation, migration and phenotype conversion of MAFB in dose -dependent manner, but the influence of MIF has reached a plateau when it's concentration was 50μg / L. TP could inhibit the proliferation, migration and phenotype conversion induces by MIF in dose-dependent manner in small doses.