| Atherosclerosis (AS) is a commen disease which causing serious harm to human health, often involving the major organ, such as heart, brain and blood vessel. It has very high morbidity and mortality, therefore prevention and treatment of atherosclerosis and improving the quality of life of patients and reducing the incidence of cardiovascular events and mortality become one of medical problems urgently needed to be solved.This dissertation includes three parts:literature research, clinical and experiment research. According to the observation to carotid atherosclerosis patients and human umbilical vein endothelial cells, the pathogenesis of AS is analysed, furthermore the effect of bushenhuoxuehuatan formula against AS and the inflammatory mechanism are discussed.Part one: literature researchThere have not the name of atherosclerosis in the ancient Chinese medical record, and generally atherosclerosis was called thoracic obstruction, angina pectoris, stroke, dizziness, headache, dementia, phlegm-fluid retention, blood stasis syndrome, etc. Zang-fu organ dysfunction, hypofunction of transportation and transformation of spleen and stomach, kidney-qi deficiency and excessive consumption of fatty and sweet foods may lead to disorder of qi activity and phlegm stagnation which gradually coming into being blood stasis. It is called syndrome of binding of phlegm and blood stasis. Many scholars believe the principal pathogenesis is phlegm stagnation and blood stasis. Professor Luo luoyi thinks that the kidney is considered as the foundation of human body and dominates the movement of qi and blood all over the body. If phlegm stagnation resulted from deficiency of kidney and disorder of qi activity, lack of warmth, transportation and transformation, blood flow slowly, stay and for blood stasis. So Professor Luo believe atherosclerosis is the syndrome of root deficiency and branch excess, the root is deficiency of kidney while the branch is binding of phlegm and blood stasis.Atherosclerosis is a chronic inflammatory disease in which cell adhesion molecules play a key role. In the early process of atherogenesis, first the endothelial cell is injured by many inflammatory factors and expression of cell adhesion molecules such as intercellular adhesion molecule-1, vascular cell adhesion molecule-1 lead to increased adherence of leukocyte. Monocytes may migrate through the endothelium and situate themselves within the subendothelial layer, engulf oxidized low-density lipoprotein, then transform in activated macrophages which continuously accumulate xidized low-density lipoprotein and become foam cells. At the same time, necrotic macrophages and proliferative smooth muscle cells result in atherosclerotic plaque. Adherence of leukocyte with endothelial cells is the initiation of atherosclerosis, whose molecular foundation is cell adhesion molecules.Part two:clinical researchObjective:To investigate the effect of Bushenhuoxuehuatan Formula on the clinical symptom score, carotid intima-media thickness, Crouse score in atherosclerotic plaque, blood lipids and the level of soluble cell adhesion molecules and explore the effect and its mechanism of Bushenhuoxuehuatan Formula on atherosclerosis.Method: 100 carotid atherosclerosis (CAS) patients were ramdomly indived into 2 groups:Bushenhuoxuehuatan Formula (BF) group and Atorvastatin Calcium (AC) group, 50 patients in each group. After two-month treatment with Bushenhuoxuehuatan Formula and Atorvastatin Calcium respectively, the clinical symptom score, carotid intima-media thickness, Crouse score in atherosclerotic plaque, blood lipids and the level of soluble cell adhesion molecules were observed before and after treatment.Results:(1) After treatment the clinical symptom score was obviously decreased in BF group and AC group (P<0.01, P<0.05). There was a significant difference between two groups (P<0.05), and compaired with AC group, BF group has better effect in improving symptom. (2) Bilateral carotid intima-media thickness was obviously decreased in BF group and AC group (P<0.01, P<0.01), and there was no significant difference between the two groups. (3) Crouse score in atherosclerotic plaque was markedly reduced in BF group and AC group (P<0.01, P<0.05). (4) The serum level of TC (P<0.05, P<0.01) and TG (P<0.05, P<0.05) in CAS patients was obviously decreased in BF group and AC group. The level of LDL-C (P<0.05) was decreased while the level of HDL-C (P<0.01) was increased in the BF group. There was significant difference in increasing HDL-C between two groups (P<0.05). (5) The serum level of soluble intercellular adhesion molecule-1 (sICAM-1) (P<0.01, P<0.01) and soluble vascular cell adhesion molecule-1 (sVCAM-1) (P<0.01, P<0.01) in CAS patients was obviously decreased in BF group and AC group.Conclusions:Bushenhuoxuehuatan Formula can be improve symptom, decrease the clinical symptom score, decrease carotid intima-media thickness, reduce Crouse score in atherosclerotic plaque, decrease the level of TC, TG, LDL-C, increase the level of HDL-C, decrease the level of sICAM-1 and sVCAM-1 in CAS patients. The effect of Bushenhuoxuehuatan Formula against AS might be related with its regulating the blood lipids and decreasing the serum level of soluble cell adhesion molecules.Part three: experimental researchObjective:To observe the effect of Bushenhuoxuehuatan Formula on the cell livability, the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and nuclear factor-kappa B (NF-κB) in human umbilical vein endothelial cells induced by tumor necrosis factor-a (TNF-a).Method: The blank serum and Bushenhuoxuehuatan Formula serum was prepared respectively by using SD rats. The primarily HUVEC isolated by 0.25% Trypsin-EDTA, then passaged, was divided into 5 groups:①Control group:HUVEC+DMEM culture medium+10% blank serum,②TNF-a group:HUVEC+DMEM culture medium +10% blank serum+10ng/mL TNF-a,③High dose BF group:HUVEC+DMEM culture medium +10% high dose BF serum+10ng/mL TNF-a,④Medium dose BF group:HUVEC+DMEM culture medium+10% medium dose BF serum+10ng/mL TNF-a,⑤Low dose BF group: HUVEC+DMEM culture medium+10% low dose BF serum+10ng/mL TNF-a. The cell livability was detected by using cell counting (CCK-8) colorimetric analysis. ICAM-1 and VCAM-1 on cell surfaces were quantified by enzyme linked immuonosorbent assays (ELISA). The expression of NF-κB was detected by immunocytochemistry analysis. The expression of ICAM-1 mRNA and VCAM-1 mRNA were examined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR).Results:(1) The cell livability was 87.2%,82% and 80% in High, medium, low dose BF group respectively,74.2% in the TNF-a group. Compared with the control group, the cell livability was decreased in other four groups, especially in the TNF-a group. (2) The expression of ICAM-1 and VCAM-1 on cell surfaces were obviously increased in the TNF-a group (P<0.01, P<0.01), compared with the control group. All doses' Bushenhuoxuehuatan Formula could inhibit the increasing expression of ICAM-1 and VCAM-1 induced by TNF-a. Compared with TNF-a group, the expression of ICAM-1 and VCAM-1 were obviously decreased in high dose BF group (P<0.01, P<0.01), and the expression of ICAM-1 was obviously decreased in medium dose BF group (P<0.05). (3) A great deal of brown-yellow particles in the cytolymph and a small quantity of deep brown particles in the cytoplasm were found in endothelial cells of TNF-a group which showed NF-κB expression was positive. Campared with TNF-a group, the positive expression of NF-κB was significantly decreased in all doses'BF group, especially high and medium dose BF group. (4) Campared with TNF-a group, the mRNA level of ICAM-1 and VCAM-1 in HUVEC was obviously up-regulated (P<0.01, P<0.01). However, high and medium dose' Bushenhuoxuehuatan Formula could down-regulate the expression of ICAM-1 mRNA (P<0.01, P<0.01), while only high dose'Bushenhuoxuehuatan Formula could down-regulate the expression of VCAM-1 mRNA (P<0.05).Conclusions:Bushenhuoxuehuatan Formula could mediate the inflammatory response of HUVEC induced by TNF-a and inhibit the expression of ICAM-1, VCAM-1 and NF-κB in endothelial cells, more significant effect in the high dose BF group. It might be the one of mechanism of Bushenhuoxuehuatan Formula against AS. |
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