Study On The Mechanism Of Anti - Atherosclerosis And Its Mechanism In

Objectives:atherosclerosis(AS), a disease of large arteries, is the primary cause of heart disease and stroke. Leonurine(SCM198), an active alkaloid typically found in Herba leonuri, was reported to have neuroprotective and cardioprotective effects, however, its effect on progressing atherosclerosis is not well known. In this study, we investigated the protective effect of SCM198on the atherosclerotic rabbits induced with high cholesterol diet (1%). Furthmore, to explore the involved pathways, we evaluated the pharmacological effects of SCM198on inflammation and oxidative stress in atherosclerotic rabbits, and further the activity of NF-κB signaling pathway. This study will provide novel evidences for research and development of new anti-atherosclerotic drugs.Methods:Part1:Fourty-two male New Zealand White rabbits (7groups, n=6) were fed a normal diet or an atherogenic diet meanwhile received treatments of low-dose SCM198(4mg/kg/day), moderate-dose SCM198(8mg/kg/day), high-dose SCM198group (16mg/kg/day), aspirin (25mg/kg/day) and placebo respectively for8weeks. Lipid parameters, whole-blood viscosity at different shear rates, plasma viscosity and K-value of ESR were measured at8weeks. Doppler echocardiographic was used to observe left ventricular function:the aortic end-diastole diameter (Dd), end-systolic diameter (Ds) and maximal intima-media thickness (IMT) were measured by high resolution ultrasonography (HRUS) performed as described before2; relative sectional change(RS) were calculated; the aortic peak velocity (Vp), mean velocity (Vm) and velocity-time integral (VTI) were recorded by Pulsed Doppler technique. The maximal systolic velocity of blood flow in the lumen of artery proximal to lesion is record to be carotid peak flow velocity (Vp)3. All the parameters were analysis in vivo vascular of atherosclerotic rabbits. At the end of the treatment, animals were sacritified, carotids and aortas were dislodged and detected. Sections were stained with hematoxylin and eosin (H&E), Oil red O staining. Endothelial cells (ECs), smooth muscle cells (SMCs) and macrophages were detected by immunostaining with PECAM-1, anti-a-SM actin and RAM11antibody. Part2:1. Malondialdehyde (MDA) content, indicating lipid peroxidation, and total antioxidant capacity were measured in serum and liver tissue. To study the mechanism, glutathione (GSH) level, activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) were determined. In aortas, the mRNA levels of antioxidant enzymes including SOD, CAT and GPx) were quantified by real-time polymerase chain reaction(real-time PCR) using SYBR Green technology.2. The concentrations of soluble ICAM-1(sICAM-1), soluble VCAM-1(s VCAM-1), IL-6and TNF-a in serums were measured by using ELISA kits for rabbits. Real-time PCR were applied to detect the relative expression of the inflammatory related genes.3. The levels of phosphorylation of NF-κB and degradation of IκB were determined by western blot.Results:part1:Compared with atheroslerotic model group, the numbers of white blood cell(WBC), red blood cell(RBC), plateles(PLT), neutrophils(Neu) and Monocytes were reduced in SCM198treated groups. SCM198showed reduction of whole blood viscosity and plasma viscosity, as well as K-value of ESR significantly (P<0.05). The concentration of high density lipoprotein cholesterol (HDL-c) in serum was significantly increased and the triglycerides (TG) was reduced in SCM198-H group(all P<0.05). Echocardiogram showed no significant difference on the related paremeters among all experimental groups. Ultrasound-derived IMT, RS and Vp in SCM198treated groups were significantly lower than those in the model group,(all P<0.05). The vascular protection effects of SCM198were further confirmed by camparation of plaque morphology during pathological examination. Fat deposition in the aorta were reduced in SCM198treated groups, macrophages formation were also inhibited by SCM198. However, no significant changes were found in smooth muscle cell proliferation in aortas of experimental rabbits. part2:1. SCM198showed a remarkable protective on the total antioxidant capacity and lipid peroxidant in serums and hepetic tissues. In the following study, we found that SCM198significantly preserve SOD, CAT and GPx activities and the content of GSH in livers. This agent also significantly upregulated SOD, CAT and GPx mRNA expression in aortic arteries.2. The level of sICAM-1, sVCAM-1, IL-6and TNF-a in SCM198treatments were all lower compared with model group (all P<0.05). Inflammatory genes, including VCAM-1, MCP-1, IL-6, TNF-a, iNOS and MMP-9, all mRNA expression levels were restored in aortas of SCM198-H treated groups in a dose-dependent manner.3. Compared with high cholesterol diet treated rabbits, the levels of phosphorylation of NF-κB in aortas were dose-dependently reduced, meanwhile, protein levels of IκB were upregulated by SCM198in a dose-dependent manner.Conclusion:1. The present study indicated that SCM198improve blood cytology, hemorrheology and fhemodynamic status;2. SCM198at high dose possessed a activity of regulating lipid metabolism in dyslipidemic rabbits via raising HDL-c and reducing TG concentration;3. The advanced formation of atherosclerotic plaque was inhibited by SCM198;4. SCM198showed anti-inflammatory and antioxidant activities on atherosclerotic rabbits;5. SCM198showed a significant potential on the inhibition of NF-KB phosphorylation and degradation of IκB.6. Taken together, SCM198has atheroprotective effect in hypercholesterolemic rabbits via suppressing inflammatory response and antioxidation, as well as the inhibition of NF-κB phosphorylation and degradation of IκB. Regulation of lipid disorder also partly involves in the anti-atherosclerotic effect of SCM198.