| Objective:1. To observe the effect of Eerdun-Wurile in atherosclerotic vulnerable plaques rabbit model;2. Explore the mechanism of Eerdun-Wurile and its optimized formula on stabilizing plaques through detecting activity of matrix metalloproteinases-1/2/9(MMP-1/2/9) and expression level.Methods:80New Zealand rabbits were divided into control group (10) and model group (70). Control group was feed regularly without surgery. Model group was subjected with high-fat diet, immune damage, balloon injury surgery to establish atherosclerotic vulnerable plaques model. After8weeks, model group was divided into model group, Eerdun-Wurile group, optimized formula group, simvastatin group randomly, and treated with drugs accordingly. After24weeks, rabbits were sacrificed, serum and aorta were collected for further study. The following items were detected.1. We tested rabbit serum TC, TG, LDL-C and HDL-C;2. We observed morphological changes of aorta by HE staining and Masson staining, measured FCT, IMT, FCT/IMT, LCA/PA and CA/PA;3. We observed the ultrastructure of aorta by electron microscope;4. We detected activity of MMP-1/2/9by ELISA;5. We investigated the expression level of MMP-1/2/9by qPCR.Results:1.57rabbits survived after24weeks treatment.9in control group,13in model group,14in Eerdun-Wurile group,12in optimized formula group,9in simvastatin group.2. Model group had higher serum fat level than control group, TC and LDL-C level in Eerdun-Wurile group and simvastatin group were lower than that in model group (P<0.05), while TG and HDL-C remained the same level. TC, TG and LDL-C level in optimized formula group were lower than model group (P<0.05), while HDL-C level remained the same level.3. Morphologic changes of rabbit aorta in each group:(1) Naked eye:Endarterium in control group was smooth and bright, the color and structure was normal, no lesion detected. Endarterium in model group was thicker and yellow-white blended, much plaque detected and constituted mountain-like structure. Endarterium in Eerdun-Wurile group, optimized formula group, simvastatin group had slight lesion, early-stage changes, plaque area (PA) was significantly lower than model group (P<0.05).(2) Optical microscope:Aorta in control group had normal structure, smooth endarterium and intact endothelial cells. Smooth muscle cells were in good shape, long or elliptic type. Cytoplasm revealed Eosinophilic red dye. These in model group revealed typical atherosclerotic changes, fibrous plaque was observed in most blood vessel, covered with thin fibrous tissue. Endothelial cells were intact or detached, many foam cells detected in unorganized pattern underneath. Decreased smooth muscle cells and proliferated SMC. Middle SMC was in chaos and submerged in inflammatory cells, couple of plaque calcified. The lesion in Eerdun-Wurile group, optimized formula group, simvastatin group was much lighter. Eerdun-Wurile group and simvastatin group had lower LCA and LCA/PA than model group (P<0.05), while no significant difference between the two. Eerdun-Wurile group and simvastatin group had higher CA and CA/PA than model group (P<0.05). Eerdun-Wurile group, optimized formula group and simvastatin group had higher FCT, IMT and FCT/IMT than model group (P<0.01). In addition, Eerdun-Wurile group and optimized formula group had higher FCT/IMT than simvastatin group (P<0.05).(3) Electron microscope:Control group had normal endarterium, flat endothelial cells, consistent cell membrane and intact mitochondria. No SMC and MNC infiltration, no foam cells formation. Model group had incomplete cell membrane and broken internal elastic lamina. Foam cells formed under endothelial cells and showed apoptotic sign. Eerdun-Wurile group showed no foam cells formation under endothelial cells, mitochondria in SMC swelled mildly. No lipid droplet in cytoplasm, no lesion in internal elastic lamina. Optimized formula group also showed no foam cells formation under endothelial cells, little lipid droplet in cytoplasm, normal endothelial cell structure in blood vessel, mitochondria swelled mildly. Simvastatin group had normal endothelial cells and fair membrane consistency. No foam cells formation under endothelial cells, mitochondria swelled mildly.4. Model group had higher serum MMP-1/2/9activity than control group (P<0.05). Eerdun-Wurile group and simvastatin group were lower than model group (P<0.05), while these two group had significant difference (P<0.05). There was no significant difference between optimized formula group and model group5. Model group had higher expression level of MMP-1/2/9than control group. Eerdun-Wurile group and simvastatin group were lower than model group (P<0.05), while these two group had significant difference (P<0.05).Conclusion:1. Multi-factor modeling is able to establish atherosclerotic vulnerable plaques rabbit model;2. Eerdun-Wurile and its optimized formula can reduce LCA, LCA/PA in atherosclerotic plaques, increase CA, CA/PA, FCT, FCT/IMT. They can stabilize plaques; 3. Eerdun-Wurile and its optimized formula can stabilize plaques through reducing serum lipid and regulating MMPs. |
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