The Mechanism Of Proanthocyanidins Anti-Atherosclerosis

Background：Atherosclerosis(AS) is the pathological foundation of ischemic heartdisease, cerebrovascular disease. Hyperglycemia, hypertension, smoking, diabetes,hyperlipidemia are the major risk factors of AS. And hyperlipidemia takes an importantpart in AS. Studies have shown that the high-fat diet can induce the AS plaqueformation in animal experiments. In the past, lipid infiltration, thrombosis and damageresponse were three theories about the pathogenesis of AS. Ross et al. firstly proposedthat AS is an inflammatory progression in1999. They considered that AS is aninflammatory reactions due to oxidative modification of ox-LDL stimulating monocytes/macrophages.The adhesion of monocytes/macrophages to vascular endothelial cells isconsidered to be the early lesions of the AS. Under the action of TNF-a, ox-LDL,monocyte chemoattractant protein (MCP-1) and so on, monocytes adhere to theendothelium and migrate to the subendothelial space. Once monocytes are in theendothelium, they differentiate into macrophages. They take up oxidized LDL(ox-LDL)through scavenger receptor and CD36and then become foam cells. The foam cells arethought to be the main component of early fatty streaks in the AS.Proanthocyanidins (OPC) is a bioflavinoid with a special molecular structure and so faris considered to be one of the most potent free radical scavenger. It has been reportedthat OPC can be used to protest against myocardial ischemia-reperfusion injury, anti-AS,reduce blood pressure, regulate blood lipids, and OPC also has a wide range ofprotective effects on the cardiovascular system. But it is still a lack of in-depth researchabout OPC against AS. In the study, we observe the impact of OPC on the foam cellsformation and explore the molecular mechanisms. Method and results:1The effect of OPC on the ox-LDL-induced macrophage-derived foamcells1.1RAW264.7cells were incubated with ox-LDL for24h to establish foam cell model.The cellular lipid accumulation was examined by oil red staining and the fluorescent ofDil-ox-LDL. Results: Under light microscope, cells were round and there was no oil-staining in the control group; cells were filled with oil-staining in the ox-LDL group,show that the foam cells formation. The color of cells in OPC group turned light and theshape became normal. OPC reduced lipid accumulation in the cells.; under a fluorescentmicroscope, compared with the model group, OPC (2,10,50μg/ml) reduced thefluorescent of Dil-ox-LDL in RAW264.7cells.1.2The expression of scavenger receptor CD36and MSR1mRNA was detected byRT-PCR analysis. Results: Compared with the control group, Ox-LDL markedlyincreased the expression of CD36, MSR1mRNA(p<0.01). While the concentration ofOPC reached50μg/ml, it down-regulated the mRNA level of CD36and MSR1(P＜0.05).2Effects of OPC on expression of ICAM-1,VCAM-1in macrophages2.1C57BL/6mice were fed with high fat diet. Normal diet group served as control.After the formation of the hyperlipidemia in12weeks, mice were randomly divided into4groups: the model control group, low dose of OPC (20mg/kg/d), high dose of OPC(50mg/kg/d/) and simvastatin group(50mg/kg/d). Mice were treated with medicine by oraladministration. After5weeks, the expressions of ICAM-1and VCAM-1on monocytes/macrophages in aortic of C57BL/6mice were detected by flow cytometry analysis.Results: The expression of ICAM-1and VCAM-1were increased in the model group,OPC decreased the expression of ICAM-1and VCAM-1on monocytes/macrophagesin aortic of the hyperlipidemia mice.2.2RAW264.7cells were incubated with ox-LDL to establish foam cell model. We detected the expression of ICAM-1and VCAM-1in RAW264.7cells by flow cytometryanalysis. Adhesion of macrophage to ECM was detected by xCELLigence System.Results: RAW264.7cells were incubated with ox-LDL (40μg/ml) for24h, the cellsurface expression of ICAM-1and VCAM-1were significantly increased;OPC(2,10,50μg/ml) decreased expression of VCAM-1induced by ox-LDL;OPC(50,10μg/ml)reduced the expression of ICAM-1induced by ox-LDL.xCELLigence SystemResults: RAW264.7cells were incubated with ox-LDL enhanced the adhesion ofmacrophage to ECM; OPC reduced the adhesion of RAW264.7cells to ECM.2.3The fluorescent probe,2’7’-dichlorodihydro fluorescein diacetate(DCFH-DA) wasused to measure the intracellular generation of ROS. The fluorescence expression ofROS was measured by flow cytometry analysis. Results: When RAW264.7cellsexposed to ox-LDL (40mg/ml) for24h, the intracellular of ROS level was significantlyincreased;Compared with ox-LDL group,OPC (2,10or50μg/ml) was able tosignificantly decreased the level of intracellular ROS.3. Inhibitory effect of OPC on the viability of RAW264.7cells byox-LDL3.1The effect of OPC on the viability of RAW264.7cells was examined by MTT assay.Results: The50ug/ml OPC incubated RAW264.7cells for24h or48h. OPC has asignificant inhibitory action on growth of RAW264.7cells induced by ox-LDL.3.2Western blotting was used to determine the expression of LC3Ⅰ/Ⅱ, ERK,p-ERK,p38and p-p38. Results: RAW264.7cells were incubated with ox-LDL, the expressionof LC3Ⅱ were increased;OPC(2,10,50μg/ml) increased expression of LC3Ⅱinducedby ox-LDL;RAW264.7cells were incubated with ox-LDL, the expression of p-ERK andp-p38have no change;OPC markedly up regulated P-ERK on ox-LDL inducedRAW264.7cells, while OPC has no significantly influence in P38leave. Conclusions1.OPC reduced lipid accumulation in the cell and reduced the foam cells formation,which was associated with the inhibition the expression of CD36and MSR1inmacrophages by OPC.2.OPC inhibited the expression of VCAM-1and ICAM-1in RAW264.7cells anddecreased adhesion capacity of ox-LDL-stimulating RAW264.7cell to ECM. OPCreduced generation of ROS on ox-LDL induced RAW264.7cells.3.OPC inhibited the viability of RAW264.7cells by inducing the autophagy.In conclusion, OPC acts against AS by decreased the foam cells formation, adhesionmolecule expression, ROS production and cells viability on the ox-LDL inducedRAW264.7cells.