Study Of Yindan Xinnaotong Capsule On Anti-atherosclerotic Effects

Research objectAtherosclerosis (Atherosclerosis, AS) is the common pathological basis of cardiovascular disease, such as angina, myocardial infarction, stroke and so on. It is one of the most serious pathophysiological process endangering life and health. And it was known as "leading killer" in developed countries and its incidence rate in our country showed a trend of rising in recent years. The pathogenesis of AS was complex, from the middle of the19th century to the1980s, Duguid, Virchow, Steinberg puts forward the theory of thrombosis, lipid infiltration theory, oxidation theory respectively to explains the formation mechanism of the AS from different angles. Ross advances injury-reaction theory which states that the arterial intimal injury was initial part of AS in1976. This theory has acquired a general common understandingat home and abroad in recent years along with the deep research of the pathogenesis of AS.From the perspective of traditional Chinese medicine, AS is an kind of systemic disease with qi and blood stasis, phlegm, meridians blockage. It is often treated with promoting blood circulation and removing blood stasis, which is a comprehensive regulative process embodying the advantages of multi-target, multi-signal pathway by Chinese traditional medicine compound. Yindan Xinnaotong capsule (YXC) is composed of Ginkgo biloba, Radix Salviae miltiorrhizae, Gynostemma pentaphyllum, Erigeron breviscapus, Allium.sativam L.var.Viviparum Regel, Panax notoginseng, Crataegus pinnatifida Bge and Borneolum syntheticum and is used as a medicine to activate blood circulation and eliminate stasis. It had a mainly effect on the treatment of cardiovascular in clinical. At present, in addition to medication and dietary control, exercise can also help to prevent AS. Exercise can regulate blood flow shear stress and vascular endothelial function and further more intervene the occurrence and development of AS.This study firstly using compound method of high fat feed, artery ligation, intraperitoneal injection of vitamin D3to establish the AS rats model. The joint of YXC and exercise were applied based on3x3factorial design, then the left common carotid artery of rat vascular pathological morphology was observe and hemorheology levels, blood lipid profile were detected to explore the joint effects of YXC and exercise in preventing AS in rats. Factors related to the vascular endothelial cells were determined and SM22alpha protein expression level was detected by immunohistochemical method to explore the mechanism of the joint effects in preventing AS. At last, in vitro the Ox-LDL, TNF alpha were used to induce human umbilical vein endothelial cells (HUVEC) injury, HUVEC survival rate was detected by MTT method after using YXC to explore the protection effect of YXC on endothelial cells. Enzyme-linked immunosorbent (ELISA) was used to determine the levels of adhesion factors, and flow cytometry instrument was used to measure endothelial cell apoptosis to explore protection mechanism of YXC. This can provide a scientific basis for the using of YXC in clinical. Research content1. The joint effects of YXC and exercise on atherosclerosis in ratsAtherosclerotic model rats were made by ligating left common carotid artery, injection of vitamin D3and feeding high-fat diet, and then3x3factorial design with two factors (YXC and exercise) was used(10groups, including9factorial design groups and1sham-treated group). After8weeks, blood samples were collected to determine blood viscosity(10s-1200s-1), plasma viscosity, fibrinogen (FIB), haematocrit (HCT), thrombin time (TT), blood lipid profile including total cholesterol (T-CHO), low-density lipoprotein-cholesterol (LDL-C), triglyceride (TG) and high-density lipoprotein-cholesterol (HDL-C). At the same time, the common carotid arteries of the rats were harvested to examine pathological changes by hematoxylin and eosin staining.2. The mechanism of joint effects between YXC and exercise on atherosclerosis in rats After8weeks, blood samples were collected nitric oxide (NO),6-keto-prostaglandin (PG) F1α, endothelin (ET) and thromboxane (TX) B2. At the same time, the common carotid arteries of the rats were harvested to examine wall thickness and vessel diameter to evaluate the effect on vascular construction remodeling of different groups. And then the expression of SM22a was assayed via immune-histochemistry to evaluate the functional remodeling of different groups.3. The study of protective function on endothelial cells by YXCHuman umbilical vein endothelial cells (HUVEC) were cultured in vitro, firstly YXC was put in to protective beforhand, then oxidized low density lipoprotein (Ox-LDL) and tumor necrosis factor (TNF-a) were used to injury, at last, cell survival rate was determinated by MTT to evaluate the protective function of YXC.HUVECs were cultured in vitro, then Ox-LDL and TNF-a were used to injury. The intercellular adhesion molecule-1(ICAM1) and platelet endothelial cell adhesion molecule-1(PECAM1) expression were detected by enzyme-linked immune method.HUVECs were cultured in vitro, then Ox-LDL was used to injury, and HUVEC apoptosis were detected by Annexin V-FITC/PI flow cytometry. Research results1. The joint effects of YXC and exercise on atherosclerosis in rats(1) The effect on AS plague Compared with sham-treated group, the vessel wall appeared to be thicker, and smooth muscle cell proliferation and lipid deposition were observed in model group. Additionally, atheromatous plaques formed, and necrosis, cholesterol crystals and calcification were present in the plaques in model group. The combination of YXC and swimming can Reduce artery blood wall calcification and decrease cholesterol crystals, the combination of2g-kg-1YXC and swimming showed a better effects.(2) The effect on hemorheology parameters In reducing plasma viscosity, blood viscosity (10s-1and200s-1), FIB, YXC and swimming showed significant interaction (p<0.05), the combination of2g-kg-’YXC and0h showed a better effects than other groups. (3)The effect on lipid profile In reducing the level of T-CHO, LDL-C, TG, the joint effect of YXC and swimming was obvious (p<0.05), YXC played the main effect on HDL-C levels(F=4.095, p=0.023).2. The mechanism of combinatiing YXC with exercise on atherosclerosis in rats(1) The effect on endothelial factor The joint effects of YXC and swimming played significant interaction (p<0.05) in increasing the level of NO,6-keto-PGF1α and decreasing the level of ET, TXB2;(2) The effect on vascular remodeling YXC and swimming have significant interaction (p<0.05)in reducing the wall thickness and wall thickness/diameter ratio; the combination of YXC and swimming can increase the expression of SM22a, the interactive effects were not significant(F=1.632, p=0.175). Swimming played the main effect on the increasing the expression of SM22a (F=8.O88, p=0.001).3. The protective effect of YXC on HUVEC induced by Ox-LDL and TNF-a(1) The maximal non-cytotoxic concentration (MNCC) of YXC was500μg·mL-1,250μg·mL-1,125μg·mL-1,62.5μg·mL-1were established as the experimental concentrations;60ng·mL-1and24h were established as the better condition of TNF-a.(2) The effects on cell survival rate YXC can obviously increase the survival rate of HUVEC induced by Ox-LDL (p<0.05) with dose-dependence; YXC can obviously increase the survival rate of HUVEC induced by TNF-a (p<0.05) and when the concentration was greater than125μg·mL-1, the dose effect relationship was remarkable.(3) The effects on adhesion molecules YXC can obviously decrease the levels of ICAM-1(p<0.05) and PECAM-1(p<0.05) of HUVEC induced by Ox-LDL; YXC can obviously decrease the levels of ICAM-1(p<0.05) and PECAM-1(p<0.05) of HUVEC induced by TNF-a.(4) The effects on cell apoptosis YXC can decrease the apoptosis rate of HUVEC induced by Ox-LDL, and inhibit the cell apoptosis.Conclusions1. The combination of YXC and swimming can prevent atherosclerosis in rats, and the possible mechanism of this was through improving hemorheological parameters and blood lipids levels, protecting the vascular endothelium and inhibiting vascular remodeling.2. YXC can protect the HUVEC injury by Ox-LDL and TNF-a, and this may be associated with the reduce of ICAM-1、PEC AM-1and the inhibitory effect on cell apoptosis.