INTRODUCTION

Xuezhikang, with HMG-CoA reductase inhibitor as the main ingredient, contains many kinds of necessary amino acids and unsaturated fatty acids. This study aims at investigating the effects of Xuezhikang on the proliferation and migration of vessel smooth muscle cells (VSMC).


MATERIALS AND METHOD

Animal Test
Thirty healthy and pure New Zealand white rabbits were chosen with body weight 2.57 ± 0.17 kg. They were divided into three groups in random. The first group was the control group taking normal feed. The second group acted as hypercholesterol group fed with normal feed plus 1.5% cholesterol. And the third group was the treatment (Xuezhikang) group taking normal feed plus 1.5% cholesterol and 0.8 g Xuezhikang per kilogram weight per day. Every rabbit was raised in a single cage with daily feed of 150 g and the treatment lasted for 12 weeks.

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 and HDL-C. Whereas LDL-C concentration was computed with the help of Friedewald Formula. At the end of 12 weeks, all rabbits were killed by bloodletting and aortic arch was taken as samples maintained 2.5% glutaraldehyde ready for the preparation of transmission electron microscopic samples.

In Vitro Study
Rabbit chest artery SMC was taken and cultivated by line-plot sticking method. Under 5% CO2 concentration, 15% calf serum RPMI 1640 culture solution was used and set still for 3-week cultivation. Then 0.1% EDTA and 0.25% pancreatic enzymes digesting solution were used to digest new SMC generations. And only well developed 3 ~ 6 generations of SMC were used in the study. After digestion, SMCs were made into 2´105/ml suspended cell solution. Then, they were added to 24-hole plate for 24-hour incubation. As soon as 24 hours passed, fresh 0.5% calf serum and RPMI 1640 culture solution with various concentrations of Xuezhikang were replaced for another 24-hour cultivation. Six hours before the reaction, each hole was added with 0.5 mg/ml 3HTdR. Cell scraper was utilized to strip cells and micro-porous membrane used to collect those cells. After that, PBS was used to wash membrane film repeatedly and the washed out was put in several bottles. Then, these bottles were placed in over under temperature of 70oC for certain period. Line 3H-TdR mixing rate of Beckman 6500 Liquid Flashing Instrument was used to determine the concentration. The cell suspended solution incubated by RPMI 1640 for 24 hours was then incubated for another 72 hours with fresh 15% calf serum and various concentrations of Xuezhikang. Finally, haemocytes counting plate was utilized to count VSMC numbers.


RESULTS

Determination Result of Serum Lipid
There was no obvious difference of the data among each group before the treatment. However, serum TC, TG and LDL-C concentrations of hypercholesterol and Xuezhikang groups rose significantly after the treatment (P < 0.05). But the above concentrations in Xuezhikang group were remarkably lower than that of hypercholesterol group
(P < 0.05). Whereas no significant differences of HDL-C level existed between hypercholesterol group, Xuezhikang group and the control group (P > 0.05). Nevertheless, a clear downturn of HDL-C level occurred in hypercholesterol group.

The Effects of Xuezhikang Concentration on the Proliferation of VSMC Under In-Vitro Conditions (See the following Table)

Transmission Electron Microscope Observation
The internal plastic plate (IPP) in the control group appeared intact, plane and even in thickness. Mid-membrane SMC contracted and inter-paralleled with IPP. Whereas underlying fissure of endothelium of hypercholesterol group expanded significantly. IPP appeared uneven in thickness and broke or even seemed like worm-bite form. Mid-membrane SMC was seen rose and intruded into underlying fissures through broken IPP. Synthesized surface form dominated SMC and endoplasmic reticulum increased and dilated. Plastiosomes increased and swelled. Meanwhile, ridges reduced or vanished or even bubbled. And lipid droplets came into being in endochylema with difference in both number and size. Partial cytoplasm lowered its density or concentrated. Myofilament dense body became vague or even disappeared. But the control group showed very little of the above changes or closed to normal.


DISCUSSION

The study demonstrated that Xuezhikang not only had effective performance to regulate lipid level, but also significantly inhibits the proliferation and migration of VSMC. This may be one of the important mechanism of Xuezhikang to prevent the formation of atherosclerosis and the re-occurrence or development of vessel restenosis after antioplasty or endangium resection.

• HMG-CoA reductase inhibitor is the speed retarding enzyme for cholesterol synthesis and mevalonic acid is the product of HMG-CoA reductase catalytic reaction. And mevalonic acid metabolism is the necessity for DNA synthesis and cell proliferation.
• Xuezhikang may directly inhibits the proliferation, migration and gene expression of SMC by suppressing mevalonic acid metabolism. Or it indirectly inhibits the proliferation and migration of SMC by adjusting lipid metabolism, improving functions of vessel endothelial cells, reducing the concentrations of plasma ET-1 and TXB2, increasing plasma PGI2 and NO and decreasing the release of platelet source growing factors.
  

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