| Objective: To investigate the body weight, serum lipid, growth factors, matrix metalloproteinases, vascular smooth muscle cells phenotype markers on established rat model of atherosclerosis and study the effects and possible mechanism of hirudo micropowder on the formation of atherosclerosis.Methods: After acclimatization for 1 week, 32 healthy male Wistar rats were randomly divided into four groups of 8 rats each namely normal control group, model control group, low and high dose groups of hirudo micropowder. The normal control group were fed with normal diet; the other groups received intraperitoneal injection of vitamin D3 at a dosage of 6×105 IU·kg-1 before a high fat diet to establish rat model of atherosclerosis. The normal control group received intraperitoneal injection of physiological saline. Since the second day, the hirudo micropowder treatment groups were intragastric administered daily with the corresponding drugs for 8 weeks. The normal and model control group received intragastric administration daily with distilled water. At the first day in fifth weeks, the groups fed with high diet received intraperitoneal injection of vitamin D3 at a dosage of 3×105 IU·kg-1 and the normal control group received intraperitoneal injection of physiological saline. The body weight of rats were recorded weekly to adjust the dosage for 8 weeks. 8 weeks later, all rats were killed and serum lipid, aortas were collected. Levels of serum low-density lipoprotein cholesterol(LDL-C), total cholesterol(TC), high-density cholesterol l(HDL-C) and triglycerides(TG) were detected. The levels of Serum platelet derived growth factor-bb(PDGF-BB) and aortic homogenate were measured by using of enzyme-linked immunosorbent assay(ELISA). The expression of α-actin and osteopontin(OPN) of vascular smooth muscle cells phenotype markers were detected by immunohistochemical staining. The pathology of aortas were observed by hematoxylin-eosin(HE) staining.Results: 1.The general observation and change of body weight in rats: At the beginning of the experiment, the rats had no significant difference in eating, activity, body weight. In model control group, one rat died with anorexia, weight loss in seventh weeks since establishing rat model of atherosclerosis. The other groups were no deaths. During the experiment, the rats in normal control group food intake is stable, body quality had been increasing. After 2 weeks of high fat diet, The model control group rats food intake were significantly decreased in different degrees and body weight were obviously slow growth and were significantly lower than the normal control group(P<0.01). The rats in hirudo micropowder treatment groups eat more volume, body weight increased more rapidly than model control group. Before the fifth weeks, the body weight of rats in hirudo micropowder treatment groups were significantly elevated than those of model control group(P<0.05 or P<0.01). Since fifth weeks, hirudo micropowder treatment group rats increased more body weight than model control group(P>0.05).2. The changes of serum lipids: In model control group, serum levels of TC and LDL-C were significantly elevated(P<0.01), TG decreased(P>0.05), and HDL-C increased(P>0.05) compared with normal control group. Compared with model control group, serum levels of TC and LDL-C decreassed significantly in hirudo micropowder treatment groups(P<0.05 or P<0.01). The levels of TC and LDL-C were lower in high dose groups of hirudo micropowder than the low dose groups of hirudo micropowder(P>0.05). The serum levels of TG increassed significantly in high dose groups of hirudo micropowder when compared with model control group(P<0.05). The levels of HDL-C were lower in hirudo micropowder treatment groups than the model control group(P>0.05).3. The changes of serum PDGF-BB: Compared with normal control group, serum levels of PDGF-BB were significantly elevated in model control group(P<0.01). The serum levels of PDGF-BB decreassed significantly in hirudo micropowder treatment groups when compared with model control group(P<0.05 or P<0.01). The levels of PDGF-BB were lower in high dose groups of hirudo micropowder than the low dose groups of hirudo micropowder(P>0.05).4. The changes of MMP-2 and MMP-9 in aortic homogenate: Compared with normal control group, the levels of MMP-2 and MMP-9 in aortic homogenate were significantly elevated in model control group(P<0.01). In hirudo micropowder treatment groups, the levels of MMP-2 and MMP-9 in aortic homogenate were significantly decreased compared with model control group(P<0.05 or P<0.01). The levels of MMP-2 and MMP-9 in aortic homogenate were lower in high dose groups of hirudo micropowder than in high dose groups of hirudo micropowder(P<0.05).5. The changes of smooth muscle cell phenotype markers α-actin in aortic arch: The positive immunohistochemical staining of smooth muscle cell α-actin were uniformity in normal control group. The levels of α-actin were significantly decreased in model control group than normal control group(P<0.05). The levels of α-actin were significantly elevated in hirudo micropowder treatment groups(P<0.05 or P<0.01). The levels of α-actin were higher in high dose groups of hirudo micropowder than low dose groups of hirudo micropowder(P>0.05).6. The changes of OPN in aortic: The OPN in aorta almost no expression in normal control group. The levels of OPN in aortic were significantly elevated in model control group than normal control group(P<0.01). The levels of OPN were significantly decreased in hirudo micropowder treatment groups than model control group(P<0.01). Inaddition, the levels of OPN were significantly decreased in high dose groups of hirudo micropowder than low dose groups of hirudo micropowder(P<0.05).7. The analysis of correlation between PDGF-BB and matrix metalloproteinases and the smooth muscle cells phenotype markers: The expression level of PDGF-BB in serum was positively correlated with the expression levels of MMP-2, MMP-9 and OPN in aorta(P<0.05) and was negatively correlated with the expression of SM-α-actin(P<0.05).8. The morphological changes of aortic arch: HE staining of aortic arch showed that aortic structure of each layer kept normal, complete, smooth, smooth muscle cells and elastic fibers arranged neatly in normal control group. Model control group aortic arch protruded into the cavity visible plaque, intimal thickening, smooth muscle cell proliferation, degeneration and necrosis occurred in the plaque, and the formation of foam cells and cholesterol crystal deposition. Aortic lesions in the hirudo micropowder treatment groups compared with the model control group had different degrees of ease. In low dose group of hirudo micropowder, the aortic arch intimal showed a small amount of plaque formation and less intimal proliferation of smooth muscle cells, plaque with a small amount of foam cells and cholesterol crystals. The aortic arch of high dose group of hirudo micropowder had small amounts of cholesterol crystal deposition, no intimal smooth muscle cells proliferation and vascular cavity had not seen the plaque forming.Conclusions:The experimental model of atherosclerosis in rats was successfully established by high fat diet combined with intraperitoneal injection of two times of vitamin D3 method. They had features such as lipid plaque formation, foam cell formation, smooth muscle cell proliferation, cholesterol crystal and so on. The intervention of hirudo micropowder on the mechanism of AS including:(1) Hirudo micropowder can improve the body weight of the rats and lipid deposition by weakening hypercalcemia on rat digestive system and vascular endothelial damage, reduce the influence of risk factors on atherosclerosis.(2) Hirudo micropowder can significantly reduce the serum TC and LDL-C levels and reduce the vascular endothelial injury and lipid deposition.(3) Hirudo micropowder can reduce serum PDGF-BB expression and downregulate the MMP-2 and MMP-9 expression of aortic, reduce the down-regulation of the α-actin of vascular smooth muscle cells phenotype markers, inhibit the expression OPN to reduce the degradation of the extracellular matrix and inhibit lipoprotein infiltration and the migration of smooth muscle cells, inhibit the phenotypic switching of vascular smooth muscle cells. Hirudo micropowder can prevent the occurrence and development of atherosclerosis.(4)Hirudo micropowder inhibits the migration and phenotypic switching of vascular smooth muscle cells may have correlation with the decrease of serum PDGF-BB expression. |
PDF Full Text Request