Atheroprotective Effect Of Probucol Is Related To Its Suppression Of Dendritic Cells

Atherosclerosis (AS) is a chronic inflammatory vascular disease involving the vascular system including coronary arteries, cerebral vessels and low extremity arteries. It causes coronary heart diseases and cerebral infarction, which affects people’s life quality. Increasing evidences has indicated that the immuno-inflammatory response plays a central role in the development of atherosclerosis. Dendritic cells (DCs) are important players responsible for the induction of immunity.Probucol, a potent anti-oxidant and lipid-lowering drug, has diverse pharmacological properties with therapeutic effects on the cardiovascular systems. It retards atherosclerosis via a range of biological activities, including its ability to exert anti-inflammatory and antioxidant activities, affect TC and HDL metabolism and maintain endothelial cell function. However, its potential anti-atherosclerotic mechanism remains to be elucidated. This study examined the hypothesis that the anti-atherosclerotic effect of probucol might be mediated by suppressing maturation of both h-monDC and DCs from murine model of atherosclerosis.In the present study, we demonstrated that probucol could suppress maturation of h-monDC induced by ox-LDL by activating HO-1. Our results also shown that in LDLR-/-mice fed a high-fat diet, treatment of probucol resulted in markedly regressed aortic atherosclerotic lesions, suppressed maturation of CD11c+DCs from secondary lymphoid organs and absent CD11c+DCs within atherosclerotic plaques. Part1Probucol effectively suppressed ox-LDL-induced and none-ox-LDL induced h-monDC maturationObjective:To investigate the effect of oxidized low-density lipoprotein (ox-LDL) and probucol on the immune maturation of human monocyte-derived dendritic cells (h-monDCs).Methods:Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coats of normal donors by Histopaque-1077density gradient centrifugation.The pure monocytes (over98%) were obtained using Anti-CD14microbeads and seeded into6-well plates, cultured in the RPMI1640medium containing100ng/ml rhGM-CSF,20ng/ml IL-4, and10%FCS. On the2nd and4th day, a half volume of the medium was replaced by the same medium. For studying the effects of HO-1, probucol and ox-LDL, HO-1siRNA and agonist were added to the DCs medium at the5th day for24hrs, then probucol (50μg/ml) was added to the DCs mediums on culture at the6th day for another24hrs and then PBS, ox-LDL (50μg/ml) were added to the medium on the7th day for another48hrs. All the cells were collected on the9th day. Cell viability as assessed by cell count and trypan blue. The immunophenotypic expressions (HLA-DR,CD86,CD40,CD1a) were analyzed by FACS and endocytosis function by FITC-dextran (1mg/mL), and cytokines secretions of culture supematants (IL-4, TNF-a) were measured with ELISA. HO-1, STAT1, STAT701protein levels were examined by Western Blotting analysis.Results:Ox-LDL promoted immune maturation of DCs:MIF of CD86.CD40, CD1a and HLA-DR was significantly increased after ox-LDL stimulation; decreased endocytosis function; increased TNF-a, suppressed IL-4(P<0.05). Furthermore, ox-LDL up-regulated STAT1701phosphorylation by activating HO-1in STAT1/CIITA. These effects were inhibited by probucol. Knocking down HO-1with specific siRNA blocked these effects of probucol.Conclusions:Probucol effectively suppressed ox-LDL-induced h-monDCs maturation via HO-1and STAT1/CIITA pathway, which may provide another way to elucidate the potential mechanism of its anti-atherosclerotic effect. PartⅡ Atheroprotective effect of probucol is related to its suppression of immune maturation of CD11c+dendritic cells in LDLR-/-miceObjective:We used a LDLR-/-mice model fed a high-fat diet to detect whether probucol also perform its anti-atherosclerotic effect on suppressing DCs maturation in vivo.Methods:Experiments were conducted with STZ-induced low-density lipoprotein receptor-deficient (LDLR-/-) mice. All the mice were fed either high-fat0.1%cholesterol-containing diet or added with0.5%probucol for4months. At the age of18weeks, the mice were euthanized; and aortic sections from the2groups were stained with en face oil red O and analyzed. The sections of aortic sinus were stained by CD11c for detecting CD11c+dendritic cells in lesion. Blood plasma was measured by the level of total cholesterol (TC) and high density lipoprotein cholesterol (HDL-C). CD11c+dendritic cells from secondary lymphoid organs (spleen and bilateral mesenteric lymph nodes) were separated. To access DCs surface marker expression, DCs were isolated with class Ⅱ major histocompatibility complex (MHC-II)-APC, CD80-FITC, CD86-FITC, CD40-PE by2-or3-color FACS analysis. Cytokines secretions of culture supematants (IL-12p70) were measured with ELISA.Results:In LDLR-/-mice fed a high-fat diet, atherosclerotic plaques were found on the luminal surface of aortas, aortic roots and opening of innominate, carotid, and left subclavian arteries. In contrast, lesions in the aortas, aortic roots and opening innominate arteries of LDLR-/-mice with treatment of probucol were obviously smaller or absent. Probucol significantly reduced TC level and increased HDL-C level in LDLR-/-mice (P<0.05). FACS analysis results showed a significant decrease in expression of CD80(63251±9221vs. 97794±1101), CD86(82525±26765vs.164460±2807), CD40(54617±2222vs.96223±15687)and MHC-Ⅱ (reduced by nearly50%) with the CD11c+DCs from probucol treated mice (P<0.05). Furthermore, we found a significant decrease in IL-12p70(21.2±7.5vs.97.1±3.0pg/ml) in probucol group (P<0.05). Our results also indicated, focal CD11c+DCs accumulation within carotid atherosclerotic plaques from high-fat group were markedly characterized by staining under confocal microscopy. In contrast, CD11c+DCs from the probucol group were nearly absent within aortic atherosclerotic plaques.Conclusions:Probucol treatment reduced atherosclerotic plaque formation in LDLR-/-mice with high-fat diet. Probucol suppressed CD86, CD80, CD40and MHC-II expression on CD11c±DCs from secondary lymphoid organs and decreased IL-12p70concentration in hypercholesterolemic mice. These results indicated that probucol significantly suppressed maturation of DCs in LDLR-/-mice on high-fat diet.