Eliminating Sputum And Removing Stasis Formulation Improves Atherosclerotic Plaque Via Regulating Mast Cell Function And Related Mechanism

BackgroundOne of the important characteristics in Atherosclerosis is all kinds of inflammation, immune cells infiltration around focus. AS one kind of secretion cell in human’s body mast cell produces its effect through degranulation and releasing a variety of active mediums and so on. Pathologic study found that there were a large number of mast cells which had been degranulated gathering the focus of coronary atherosclerosis plaque rupture which suggested mast cells might closely associated with the development process of atherosclerosis plaque. Traditional Chinese Medicine believes "phlegm stasis mutual knot" is the mechanism of AS. In the theory of Traditional Chinese Medicine the embodiment of "phlegm block " is the generating of lipid streaks and the foam cells gathering beneath the endarterium. Platelet activation and high coagulation state represent " blood stasis syndrome ".The Eliminating Sputum and Removing Stasis Formulation (ERF) developed by professor zong-gui Wu had been proven to improve New Zealand rabbit hyperlipidemia and regulate mice cell immune function. Now we want to study how Eliminating Sputum and Removing Stasis Formulation improves Atherosclerotic Plaque via regulating Mast Cell function and related mechanism in the apo E-/- mice atherosclerotic plaque model.Methods1. Establishment and evaluation of artery atherosclerotic plaque model on apoE-/-mice: we choose 6-8-week-old male ApoE-/- mice as the model animal.(1)Establish the atherosclerotic plaque model. All Mouse were received partial left carotid ligation and left kidney artery stenosis operation. Supply high-fat diet to mice until the end. (2) Evaluation the effect of model. We measured mice’s carotid artery blood pressure via the method of carotid artery intubation and the blood flow velocity of carotid artery by Doppler ultrasound after modeling four weeks later. We would also compare carotid artery pathological section to evaluate the situation of modeling follow-up.2. Discussing the preliminary mechanism of Eliminating Sputum and Removing Stasis Formulation that how does it improve atherosclerosis plaques via regulating mast cells’function. We divided mouse into four groups randomly:control group(CON), model group(MOD), atorvastain group(ATO) and Eliminating Sputum and Removing Stasis Formulation group(ERF). n=10. Each group fills different laboratorial drug into their stomach in line with plan every day. After twelve weeks all mouse were anesthetized and killed by euthanasia. We compare plaque area and plaque inside lipid load area by aortic intima and aortic root frozen section oil red o staining respectively. Testing mice plasma lipoprotein by fast protein liquid chromatography (FPLC). The level of total cholesterol and total triglyceride was tested by related reagent kit (enzymic method).The Serum tryptase activity tested by Colorimetric method. Comparing all groups’ mast cells activation genes expression and serum inflammatory cytokines by Quantitative PCR and Western blot. The leves of mast cell degranulation among different groups through toluidine blue staining on the aortic root slicing.Result1. Evaluation of atherosclerotic plaque model based on ApoE-/-mice. (1).Systolic blood pressure was tested by the tube input into the carotid artery of ApoE-/- mice. Through analysis of statistics the results were that the average systolic pressure of control group was 108.9±9.8mmHg and other model groups were 154.5±9.1mmHg, prompting the success of operation of stenosis on left kidney artery. (2). Evaluation on Doppler ultrasound equipment on blood flow of mice. We test the blood flow of left carotid artery by Doppler ultrasound, finding compared with control group (627.52±44.82mm/s), model groups in 4 weeks were 205.72±36.84mm/s and the peak systolic velocity (Vmax) drops dramatically (P<0.05), the difference of value of Vmax among the groups makes no sense statically (P>0.05) (3). Evaluation on section staining of carotid artery. Compared with control group(539.32±58.92 ×103um2), ligation of carotid arteries in model group produced seriously atherosclerotic plaques (790.59±92.12×103um2). The results prompt low shear stress in the carotid artery and high blood pressure led to form and develop about atherosclerosis.2. Discussing the preliminary mechanism of Eliminating Sputum and Removing Stasis Formulation that how does it improve atherosclerosis plaques via regulating mast cells’function. Among groups ERF is the most significance on lower the level of mice plasma TC (P<0.05). The level of Low Density Lipoprotein(LDL) and activity of Tryptase are all descend remarkably (P<0.05). The mast cell secretion related genes in aortic vascular tissue and serum inflammatory cytokines such as Leukotriene C4synthase (LTC4S)、Histidine decarboxylase (HDC)、mast cell chymase (CMA1)、Mast Cell Tryptase 6 (MCT6) and Tumor Necrosis Factora(TNF-α)、transforming growth factor 1 (TGF-Pi) exist difference dramatically among ATO and ERF (P<0.05). Meanwhile we observe from the Aortic vascular root section oil red o and toluidine blue staining finding that the model can accelerate the artery plaques formation. Eliminating Sputum and Removing Stasis Formulation can improve atherosclerotic plaques via reducing arterial plaques area, reducing lipid load level in the plaques, weaken the degree of mast cell aggregation and degranulation (P< 0.05).Conclusion1、Through partial left carotid ligation and left kidney artery stenosis operation and high-fat diet apoE mice can successfully be induced atherosclerotic plaque formation.2、Eliminating Sputum and Removing Stasis Formulation and atorvastatin have the similar effect on the aspects of decreasing arterial plaques load area, reducing lipid load in the plaques and lower plasma TG and LDL levels.3、Compared with atorvastatin, The Eliminating Sputum and Removing Stasis Formulation may have extra effects on reducing the content of TC, weakening mast cells aggregation and degranulation on outer membrane artery and adjusting the level of TNF-α、TGF-βlin the blood.