The Effect Of Danlou Tablets On The Carotid Atherosclerotic Plaque In Rabbits

The number of patients with atherosclerosis (AS) is rising quickly, which imply hugeburden on the society and family. Atherosclerotic associated disease is the most commoncause of mortality and morbidity of patients in developing and developed countries. Thestability of carotid atherosclerotic plaques determines the risk of plaque rupture, whichmay lead to thrombus formation and potentially severe clinical complications such asmyocardial infarction and stroke. Therefore, to increase the stability of plaque is veryimportant. Furthermore,the traditional Chinese medication treatment which was shownlittle or no side effects, might offer a great potential value for plaque stabilization. Danloutablets include pericarpium trichosanthis, savia miltiorrhiza, paeonia veitchii, xiebai,chuanxiong and et al. Such drug has vertified to sooth the chest oppression to promoteyang, remove the phlegm and turbid urine, and activate blood circulation to dissipate bloodstasis. This study was carried out to observe the effects of Danlou tablets on establishedvulnerable carotid plaques in rabbits and invovled mechanisms.Materials and Methods:1. Rabbit model of vulnerable plaques: A total of70male New Zealand White rabbitswere fed with high-cholesterol diet (1%cholesterol) for8wk，the left carotid arteryadventitia of rabbit was injured with digestion and blunt dissection, meanwhile thecontralateral one obtained sham operation. Rabbits were randomly divided into fivegroups for16-wk treatment, control group that received no treatment (the regressiongroup), low-dose DL group, moderate-dose DL group, high-dose DL group, andatorvastatin group.2. Blood lipid measurement: Blood was drawn from ear vein of rabbits fasting overnightat baseline and at the end of weeks8and24. Serum levels of total cholesterol (TC),triglycerides (TG), lipoprotein, low-density lipoprotein cholesterol(LDL-C) andhigh-density cholesterol (HDL-C).3. High-frequency ultrasonography assay: At the baseline and at the end of weeks8and24, a high-frequency duplex ultrasonographic system was used to acquire images of thecarotid artery from all rabbits starved overnight. We used Vevo770system tomeasure atherosclerotic plaques in the baseline ultrasound parameters. By carotidultrasonography, carotid artery intima thickness, the plaque site, size and echocharacteristics were observed. plaques in thecarotid artery long axis and short axis via two-dimensional echocardiography were obtained.4. Histopathological analysis：Arteries were collected for paraffin sections. Paraffinsections were assessed by hematoxylin&eosin (H&E) and immunohistochemicalstaining. The carotid aretery intimal hyperplasia was identified by a computer-assistedimage analysis system. Immunohistochemical staining was used to analyze cellularcomponents of intimal lesions.5. Inflammatory cytokines Analysis: The serum levels of Adiponectin, interleukin-6(IL-6), tumor necrosis factor-α (TNF-a), interleukin-6(IL-10) and oxidized LDLcholesterol (ox-LDL) were measured by use of enzyme-linked immunosorbent assay(ELISA).6. RT-PCR：The mRNA expressions of MCP-1、ICAM-1and TIMP-1in the carotid arterytissue were analyzed using RT-PCR technique．Results:1. Lipid measurement: Serum levels of TC, TG, LDL-C and HDL-C at baseline in allrabbits with no significant differences among the five groups of rabbits(P>0.05). TC andLDL-C were significantly elevated in all five groups of rabbits after a high cholesterol dietfor8wk（P<0.001）.In the DL and atorvastatin group, the levels of TC and LDL-C were significantly reducedat weeks24compared with the regression group (all P <0.001). DL administration withlow and moderate doses all significantly lowered serum levels of TG compared with theregression group (all P<0.01), but did not differ significantly between high-dose DL groupand high-dose atorvastatin group(P>0.05).2. High-frequency ultrasonography assay:Values of LCAAD and Square weresignificantly lower in atorvastatin group and DL with moderate-dose and high-dose groups,than in the regression group (P<0.01~0.05). The values of Vmax and RI did not differamong all five groups (P>0.05).3. Histological and immunohistochemical staining analysis: The atherosderotic lesionof left carotid was more serious than the right side among the five groups, the right(sham operated side) with only mild hyperplasia. Immunohistochemical staining detectedthat both macrophages and smooth muscle cells presented in plaques.4. Inflammatory factors detection: The serum levels of IL-10and Adiponectin werehigher in the treatment groups compared with the regression group（P<0.01~0.05）.However, the levels of MCP-1,IL-6, TNF-α, and ox-LDL were lower in all treatment groups than in the control group significantly（P<0.01~0.05）.5. RT-PCR：The mRNA expressions of MCP-1and ICAM-1in the atherosclerosticlesions of the carotid artery in DL group and atorvastatin group all significantly loweredcompared with the regression group (P<0.01or P<0.05); The mRNA expressions ofTIMP-1in the atherosclerostic lesions of the carotid artery was higher in the treatmentgroups compared with the regression group（P<0.01~0.05）.Conclusions:The rabbit model of vulnerable plaques call be established efficiently byhigh-cholesterol diet and vascular membrana adventitia injury． Danlou tablesdose-dependently increased the stability of the vulnerable plaques and prevents plaquesfrom rupture. Atorvastatin and the Danlou tables offer a similar protective effect vialipid-lowering, anti-inflammation and antioxidation effects. But high-dose DL tablesregress the atherosclerotic vulnerable plaques demonstrated the unique role, which may berelated to elevated serum levels of HDL levels is closely related.