INTRODUCTION

The formation of atherosclerosis results from a series of inflammation due to endothelial damages caused by many coronary heart disease inducing factors.

• Among them, malfunctions of endothelial cells, i.e. the disturbed balance between NO, ET, PGI2 and TXA2 produced and released by endothelial cells (EC) is regarded as the key and initial driving factor of the process.
• Xuezhikang is a highly effective lipid regulating medicine made and refined from traditional Chinese medicine red yeast rice (Monascus purpureus). It has HMG-CoA reductase inhibitor (HMG-CoARI) as its main ingredients together with many kinds of necessary animo acids and unsaturated fatty acids. In this study, 1.5% cholesterol was used to establish rabbit atherosclerosis model in order to observe the protective effects of Xuezhikang on functions of endothelial cells in cholesterol-fed rabbits.
  
  

METHODS AND MATERIALS

The Establishment of Rabbit Atherosclerosis Model
Thirty healthy pure Newzealand Rabbits were used in the study, 15 male, 15 female, weight 2.57 ± 0.17 kg, 4 months old. Tested rabbits were raised for adaptation for 2 weeks before the study and all those abnormal in diet were excluded. The ones with stable lipid level and metabolism were randomized into three groups. The control group was fed with ordinary granular feed, hypercholesterol group fed with common feed plus 1.5% cholesterol and Xuezhikang group with common feed plus 1.5% cholesterol and 0.8 g/kg·d of Xuezhikang. Xuezhikang was the product of Peking University WBL Company. Every rabbit was raised in single cage with 150 g food per day and water available. The study lasted for 12 weeks..

Measurement and Instruments
The determination of TC, TG and HDL-C concentrations: it was carried out by enzyme method using reagent cartridge developed by Beijing Zhongsheng Bio-Tech Company and Hitachi Model 7060 Automatic Biochemical Analyzer was used for the test. And the concentration of serum LDL-C was calculated by the following formula:
LDL-C = TC - HDL-C - TG/2.2

The determination of plasma ET-1, PGI2 and TXA2: reagent cartridge manufactured by Beijing Dongya Immune Technology Research Institute was used. Automatic counter was used to conduct imbalance radioimmunassay so as to detect the concentrations of ET-1, 6-keto-prostaglandin (PG) F1a and TXB2.

The determination of trace serum nitrous oxide[3,4] : imbibe 0.5 ml serum, add 0.1 ml 35% sulfosalicylic acid precipitated protein and centrifugalize them (-4oC, 1000´g,
15 minutes). Take 0.1 ml up-layer clear solution, add 0.1 ml Griess reagent and 0.1 ml 4 mol/L HCL to react under room temperature for 10 minutes. Use enzymo-immunometer to read absorbance at 570 nm. Employ nitrite for standard curve and test results are expressed in nitrite mg/L transformed from NO.

Material, Preparation and Observation of Pathologic Sample
In the morning, blood samples were taken from ear mid-artery on fast animals before, 4 weeks, 8 weeks and 12 weeks after the study to determine the concentrations of serum TC, TG, HDL-C, and plasma ET-1, 6-keto- PGF1a and TXB2. At the end of 12 weeks, all rabbits were killed by bleeding. The Artery were taken from the very root to arteria iliaca communis branch. External membrane and connective tissues were stripped. It was cut along abdomen side and then cleaned by 0.9% normal saline. After that, aortic arch and musculus cardiacus samples were taken. They were maintained in 10% methanol and 2.5% glutaraldehyde. Routine procedures were followed to make microscopic and transmission electron microscopic samples so as to monitor histological changes of aortic arch, coronary artery and vessel endothelial cells (VEC).

Statistics Methods
All data were expressed in average value ± standard derivation (x ± s) and t-test used between the average value of two groups. However, multi-group data comparison was analyzed by variance analysis. While linear correlation analytical method was used for different indexes.

RESULTS

Effects of Xuezhikang on Serum Lipid (Table I)
There were no distinct differences between the groups before the study. However, at 4 weeks, 8 weeks and 12 weeks after treatment, the concentrations of serum TC, TG and LDL-C of hypercholesterol and Xuezhikang groups were dramatically higher than that of the control (P < 0.05). While that of Xuezhikang group were clearly lower than that of hypercholesterol group (P < 0.05). However, HDL-C level of both hypercholesterol group and Xuezhikang group had no significant difference compared with that of the control (P > 0.05). But there was a clear downturn of HDL-C level in hypercholesterol group.

The Effects of Xuezhikang on Plasma ET-1 and Serum NO
It could be seen from Table II that no obvious differences exist among data of each group before the treatment. However, 4 weeks, 8 weeks and 12 weeks after treatment, plasma ET-1 level in hypercholesterol group and Xuezhikang group was remarkably higher than that of the control group. Whereas serum NO was lower than that of the control group (P < 0.05). Meanwhile, ET-1 of Xuezhikang group was lower than that of hypercholesterol group while NO was higher (P < 0.05).

The Effects of Xuezhikang on Plasma TXB2, 6-keto-PGF1a and TXB2/6-keto-PGF1a (Table III)
There were no significant difference between the groups before the treatment. However, 4 weeks, 8 weeks and 12 weeks after the treatment, plasma TXB2 and TXB2/6-keto-PGF1a ratio of hypercholesterol group were significantly higher than that of the control group (P < 0.05) while 6-keto-PGF1a was lower but with no statistic significance. There was no obvious change of plasma 6-keto-PGF1a level in Xuezhikang treatment group. The concentration of TXB2 had a remarkable increase, but still significantly lower than that of hypercholesterol group (P < 0.05). Meanwhile, TXB2/6-keto-PGF1a ratio began to rise 8 weeks after the treatment, but still dramatically lower than that of hypercholesterol group (P < 0.05).

Analysis of Correlation between Serum Lipid and ET-1, TXB2 Levels as well as TXB2/6-keto-PGF1a Ratio
It was found that plasma TC, LDL-C and TG were positively correlated with ET-1 and TXB2/6-keto-PGF1a ratio (r = 0.762 ~ 0.957, P < 0.001). Whereas ET-1 had positive correlation with TXB2/6-keto-PGF1a ratio (r = 0.921, P < 0.001).


Pathomorphology Observation
Aortic intima of the rabbits in the control group was smooth and plane. The mid-membrane smooth muscle cells (SMC) positioned regularly and alternated with plastic plate in approximate parallel form. Large amount of foam cells occurred at the end of 12 weeks underneath aortic intima in the hypercholesterol group. There were about 9 ~ 14 layers with some foam cells broken and lipid kinds of materials free outside cells. In addition, the mid-membrane smooth muscle cells (SMC) positioned irregularly and internal plastic plate disassociated and broke. And the trend of SMC migrating to intima was seen. The intima of pro-descending section of coronary artery increased its thickness and partial myocardial micro-vessel were almost blocked. In the Xuezhikang group, however, aortic artery pathological changes of the control group were much less than that of hypercholesterol group. There were only 4 ~ 5 layers of foam cells and coronary artery was nearly normal. There were only few foam cells at the branch. Under electron microscope, it could be seen that endothelial cells of the control group were flat and plane, EC closely connected, fissure under endothelium being narrow and various levels of organoids such as endoplasmic reticulum and plastiosome appeared in cytoplasm. At the end of 12 weeks study, endothelial cells of hypercholesterol group changed the nature and swelled, inter-EC connection obviously expanded and various number of lipid droplets occurred in cytolymph. Moreover, cyton intruded into envelope or dropped. However, the micro-structure of endothelial cells in Xuezhikang group behaved normally.


DISCUSSION

The assumption that damage of vessel endothelial cells can lead to atherosclerosis was firstly presented by Ross.[1] Endothelial cells are located between vessel wall SMC and circulating platelets as well as mononuclear cells. With mechanic and humoral stimulation, EC deactivated vessel active materials such as serotonin and delayed stimulate peptide, released vessel contraction materials (ET-1, angiotensin II) and vessel expansion materials (PGI2, NO). Thus regulating vessel tensile force, the adhesion and concentration of platelets and monocytes, as well as improving the balance of coagulation and cellulolytic process. Therefore, it is of great significance to protect vessel endothelial cell functions in order to prevent and control atherosclerosis.

Hyperlipoidemia is one of the causes of various kinds of endothelial cell damage. This study demonstrated an increasing dramatic increasement of plasma ET-1, TXB2 and TXB2/6-keto-PGF1a ratio and a fall of NO in hypercholesterol group rabbits 4 weeks after high cholesterol feed. Although there were no significant statistic difference in 6-keto-PGF1a level between Xuezhikang group and that of the control group. However, the concentration of 6-keto-PGF1a went down as TC increased. After 12 weeks treatment, aortic arch and coronary artery in hypercholesterol group had formed obvious pathological changes of atherosclerosis. Under electron microscope, vessel endothelial cells appeared cytometaplasia and cyto-swell and the change in microstructure was rather obvious. Therefore, it could be seen that at early stage of feed induced atherosclerosis in rabbits, vessel endothelial cell functions changed and went worse with time. In Xuezhikang group, however, though there were some changes in the concentrations of ET-1, TXB2 and TXB2/6-keto-PGF1a ratio, they were much slighter than that of hypercholesterol group. (P < 0.05). And the number of atherosclerosis scar was significantly less than that of hypercholesterol group. Meanwhile, micro-structural changes appeared slighter. So, it demonstrated that Xuezhikang could delay the increase of plasma ET-1 and the decrease of NO resulting from hypercholesterol diet, maintain the balance between TXB2 and 6-keto-PGF1a level, thus protecting vessel endothelial cell functions and inhibiting the formation of atherosclerosis.

Under normal conditions, ET-1 is produced and secreted from vessel EC. It is the strongest vessel-contracting active polypeptide [5] ever found. It acts as mitogen by activating phosphatidase C, stimulating c-fos and c-myc gene expression of vessel SMC (VSMC), increases the synthesis of DNA of VSMC and promoting the proliferation of VSMC. [6,7] Therefore, it plays a leading role in the formation of atherosclerosis. NO is produced from vessel endothelial cells under physiological conditions. By activating guanylic acid cyclase, it increases cGMP in cells and leads to the following effects: relaxing VSMC; maintaining vessel expansion; inhibiting platelet adhesion and concentration; affecting the activity and expression of white cell adhesive molecules CD11 and CD18, suppressing cell division and proliferation of SMC; reducing the production of collagenous fibres and plastic fibres; taking additional electrons; cleaning free radicals and inhibiting peroxidation of lipids. [8,9] Thus, protecting normal endothelial cell functions and maintaining the balance between serum ET-1 and NO, which are produced from endothelial cells, is one of the key mechanisms of Xuezhikang in suppressing the formation of atherosclerosis.

Many studies showed that the imbalance between TXA2 and PGI2 is one of the causes of the incidence of atherosclerosis. The imbalance in this study results from the increase of TXA2. The main reasons are: the development of atherosclerosis resulting from hypercholesterol diet causes the production of lots of lipid peroxidants (LPO) that can directly damage EC, inhibit prostacyclin reductase, reduce the synthesis and release of PGI2, and in the mean time promote the production of platelet TXA2. In the case of hyperlipoidemia, ET-1 concentration goes up significantly. It can activate phosphatidase A of VMSC, lead to the release of arachidonic acid, strongly stimulate the formation of TXB2 and prostaglandin, activates ET B receptor on EC surface and promote the release of PGI. When endothelial cells damage, the release of platelet increases causing the release of large amount of TXA2. Meanwhile, cholesterol can directly act on platelet leading to increased release of TXA2. This study shows that Xuezhikang can correct the imbalance between TXB2 and 6-keto-PGF1a. Also, serum TC, LDL, ET are positively correlated with TXB2/6-keto-PGF1a. And Xuezhikang can lower TXB2/6-keto-PGF1a ratio through regulating lipid metabolism and inhibiting the secretion of ET-1. In addition, it can also inhibit the production of lots of lipid peroxidation. The increase of vessel wall PGI2 may account the main reason.

Functional changes of endothelial cells may occur at the early development of atherosclerosis, even before the formation of scar or clot. A series inflammable reaction resulting from endothelial cell damage facilitate the development of atherosclerosis. [1,4]
The study verifies that Xuezhikang has protective effects for rabbit endothelial cell functions fed with high cholesterol diet.

REFERENCES