Development Of Atherosclerotic Plaque In ApoE^-/- Mice And The Effect Of Paeonol Intervention

Atherosclerosis（AS） is a common vascular lesions disease which is seriously harmful to human health. According to the development of the pathological changes,AS can be classified as fatty streaks and lipid plaque, fibrous plaque, atheromatous plaque and the secondary changes of atheromatous plaque. Lesions in the initial stage,because of the role of promoting atherosclerosis factor, cause oxidative stress and inflammation.Thus inducing adhesion of monocytes on the monolayers of VECs,forming early fatty streaks spot. Mononuclear cells further into vascular intima, divided into a foam cells. In addition, proliferation of vascular smooth muscle cells were improved, two kinds of foam cell formation of fibrous plaques together. As the disease progresses, the plaque surface is made up of elastin and fibers of collagen, including macrophages, cellular debris, and lipid accumulation of mi atherosclerosis material,commonly known as atheromatous plaque. Dyslipidemia, inflammation and oxidative stress are play an important role in the whole process of occurrence and development of AS.Paeonol（Paeonal, Pae） is one of the active ingredients dried root bark of Paeonia Suffruticoas Andr. and the dried root and rhizome of Cynanchum Cynanchum paniculatum（Bge.） Kitag, possessing the pharmacological effects of anti-arrhythmic,anti-hypertensive, anti-thrombotic cardiovascular system, such as a wide range. Modern pharmacological studies on pae indicate that it could anti-inflammatory, lipid-lowering,anti-atherosclerosis, antithrombotic and other pharmacological effects, etc.ApoE^-/- mice as the ideal animal model, but its occurrence and development of blood vessels of atherosclerosis plaque process and Pae reported for the first study of its impact, it remains to be further explored.OBJECTIVEIn this study, through the establishment of ApoE^-/- mice model of atherosclerosis.Objective to observe the characteristics of stage of fatty streaks, fiber plaque of atherosclerosis, atheromatous plaque, the changes of blood-lipid, oxdative stress,inflammatory indicators. Enunciating the characteristics of the antiatherosclerotic mechanism, and providing theoretical basis for deep study.METHODSTo establish the ApoE^-/- arteriosclerosis model with a high-fat diet.Using H&E, oil red O, Masson staining to observe different pathological changes of atherosclerosis aortic pathological morphological and the intervention effect of Pae.Their serum Total cholesterol（TC）, triglycerides（TG）, HDL-C, LDL-C levels were measured with automatic chemistry analyzer. ELISA was used to measure the levels of Interleukin-6（IL-6）, TNF-α, ox-LDL in Serum. At the same time using Western Blot method to detect the expression of MMP-9 and VCAM-1. TAB method to detect the contents of MDA. WST-8 method to detect SOD’s vigor and Pae’s intervention effect.RESULTS1 The establishment of ApoE^-/- mice atherosclerosis model and different characteristic lesions in period of change1.1 ApoE^-/- mice artery atherosclerotic lesions in different period of aortic pathology morphological changesRandomly selected a batch of ApoE^-/-mice, according to the weight in three groups:group a（M-a） and group b（M-b） and group c（M-c）, feeded high-fat for 5, 16 and 24 weeks. Compared with the control group, high-fat feeding at 5 weeks（M-a）, the earliest time examined, mononuclear cell adhesion to the endothelium was observed in the ApoE^-/- mice. The spindle-shaped cells were intermingled with the foam cells or tended to form a cap at the top of the lesion. By 16 weeks later（M-b）, early fibrous plaques were observed in ApoE^-/- mice on the high-fat diet. These lesions contained small necrotic cores together with a few foam cells that were covered by a well-formed fibrous cap containing smooth muscle cells surrounded by elastic fibers and collagenous tissue. In older mice, the fibrous plaques appeared more advanced, with larger necrotic cores and abundant fibrous tissue. Electron microscopic analysis of these lesions revealed large, lipid-laden, macrophage-derived foam cells together with numerous lipid-containing smooth muscle cells, identified by their myofilaments, dense bodies,and surrounding basement membrane.1.2 The change of serum lipidResults show that, compared with control group, the ApoE^-/- mice atherosclerosis in different periods of blood lipid levels were significantly increased.1.3 Serum TNF-α, IL-6 expression levelsCompared with the control group, until for 16 weeks, serum TNF-α was significantly higher, the expression increasing with pathological changes level with statistical significance. At 5 weeks, serum IL-6 was significantly improved.1.4 The protein expression of aortic VCAM-1, MMP-9Compared with the control group, protein expression of aortic VCAM-1, MMP-9were significantly increased. Compared with the fatty streaks period, protein expression of aortic VCAM-1, MMP-9 were significantly increased in M-b and M-c.1.5 Serum ox-LDL, SOD and MDACompared with the control group, serum ox-LDL, MDA were significantly increased.When atherosclerosis development into atheromatous plaque ox-LDL and MDA increased significantly. Serum SOD activity was significantly decreased.2 The treatment of Pae on atherosclerosis in ApoE^-/- mice2.1The effect of Pae in ApoE^-/- mice aorta lipid accumulation and plaque formationModel a, b and c feeded high fat for 5, 16 and 24 weeks, Each group has given to different doses of Pae（400, 200 and 100 mg·kg-1）. After 5 weeks of treatment with Pae,there was a significant improvement in aortic lesions.2.2 The effect of Pae on lipid levelsResults show that, after 5 weeks of treatment with Pae, there was a significant decrease in plasma lipids.2.3 The effect of Pae on expression of serum TNF-α, IL-6Results show that in model b, c: compared with the control group, TNF-α was significantly increased, compared with model group, Pae inhibited the expression of TNF-α, and present a dose-effect relationship. Model a, b and c: compared with the control group, IL-6 was significantly increased, compared with model group, Pae（400mg·kg-1） inhibited the expression of IL-6.2.4 The effect of Pae on protein expression of aortic VCAM-1, MMP-9Compared with model group, Pae（400, 200 and 100 mg·kg-1） cut the expression of VCAM-1 after the treatment, and there are dose dependent. Model a: the Pae group（400 mg·kg-1） cut MMP-9 protein expression, Pae group（200 and 100 mg·kg-1） there are no significant difference compared with model group. Model b,c: compared with model group, Pae（400, 200 and 100mg·kg-1） can significantly lower the MMP-9 protein expression.2.5 The effect of Pae on serum ox-LDL, SOD and MDAResults show that the model b, c: compared with the control group, ox-LDL expression level increased significantly, Pae inhibited the expression of ox-LDL significantly. Model a: treatment with Pae, there are no significant difference compared with model group; Model b, c: Pae（400, 400 and 200 mg·kg-1） significantly increased SOD vigor. Model a: compared with model group, treatment with the Pae（400mg·kg-1）MDA content decreased significantly, Model b, c: compared with model group,treatment with the Pae（400, 200 and 100 mg·kg-1）, MDA was decreased significantly.CONCLUSION1. After 5, 16 and 24 weeks on lipid rich food, the ApoE^-/- mice atherosclerosis disease development and enter the stage fatty streaks, fibrous plaques and atheromatous plaque, increased with the cycles, the artery plaque lesions has improved, lipid accumulation has increased. In the atherosclerosis disease progress in different period,lipid levels were significantly increased; After the formation of fibrous plaque, serum TNF-α was significantly higher; After the occurrence of atherosclerosis serum IL-6 and protein expression of aortic VCAM-1, MMP-9 were significantly increased, the expression increasing with pathological changes level with statistical significance;serum MDA, ox-LDL were significantly higher; serum SOD activity was significantly decreased.2. The Pae have certain relief effect. It have played an important role in the treatment of AS maybe by lowering the serum TC, TG and LDL-C levels. In fibrous plaques and atheromatous plaque period, Pae possesses anti-oxidative stress and inflammatory response, thereby it can protect the vascular endothelium and play a therapeutic role in atherosclerosis.