| Objective:To study the treatment effect of TCS on AS and its mechanism and to provide a theoretical basis for resource utilization of semen celosiae and development new drugs anti-atherosclerosis.MethodsIn vivo:To evaluate atherosclerosis, ApoE-/- mice were fed western high fat diet for 12 weeks. Set three TCS doses:10mg/kg,30mg/kg,90mg/kg, administered four weeks and then observed the effects on relative area and stability AS and lipid plaque in mice.In vitro:Ox-LDL stimulated peritoneal macrophages 24h to establish an in vitro model of AS. On this basis, we evaluated TCS anti-AS effect by researching peritoneal macrophage phagocytosis, formation of foam cell, genes associated protein expression and autophagy on stream to explore its pharmacological mechanism.ResultsTCS anti-AS pharmacodynamics:TCS 10 mg/kg～90 mg/kg significantly reduced the prevalence of the relative area of plaque in ApoE-/- mice aorta, and showed the therapeutic effect on AS. More details as follow:1. Oil red O staining for aortic overall:in model group, there were a large number of plaques in the aorta. The plaque on upstream is large area and protruding obviously, even downstream a certain amount of plaque existet stubbornly. After administrated with TCS, aortic plaque reduced in different degree almost invisible in the downstream.2. Oil red O staining cross section of aortic root:in model group, amount of lipid was accumulated on the aortic lumen wall. The plaque was thick and large relative area. While after the administrated of TCS, the accumulation of lipid on the inner wall of aortic was reduced significantly and the relative plaque area was reduced. Particularly, part area of aortic inner wall, in the TCS 30.0 mg/kg and 90.0 mg/kg groups, was smooth and no obvious fat accumulation and plaque presence.3. H&E stanning for cross section of aortic root:in model group, the relative plaque area was large and necrotic core presented as eccentric, and a large accumulation of cholesterol. Compared with model group, the relative plaque area was smaller and less accumulation of cholesterol.4. Sirius red stanning for cross section of aortic root:in model group, there was little total collagen in plaque area and small ratio type I/type III, indicating that the plaque was vulnerable. Compared with model group, there was more total collagen in plaque and larger ratio type Ⅰ/type Ⅲ, indicating that the plaque was more stable.5. Immunofluorescence for cross section of aortic root:compared with model group, the number of autophagy bodies in TCS group increased significantly, suggesting that anti-atherosclerosis effect of TCS may be related to its promoting autophagy.TCS anti atherosclerosis mechanism:In vitro, TCS reduced the phagocytosis of macrophages on lipid. Compared with model group, macrophage phagocytosis rate of lipid decreased to 53.0% in TCS 50.0 g/mL group. In addition, TCS 12.5 μg/mL-50.0 μg/mL were also reduced the formation rate of foam cells (P<0.05 or P<0.01) and the content of TC (P<0.01). The mRNA expression of CD36 and SR-A1 were down-regulated by 56.5% and 62.4% respectively, while the mRNA expression of ABCA1 and ABCG1 were up-regulated by 3.1 times and 1.9 times. To terms of protein, expression of LC3 and beclinl were increased within the administrating of TCS 12.5-50.0 μg/mL, suggesting that TCS can promote the increase of autophagy. Intuitively, under the electron microscope, the number of autophagy bodies in TCS (50.0 μg/mL) group was significantly higher than that in the ox-LDL stimulated group. Further, the ratio of the lysosomal/autophagy body was significantly higher than that of the ox-LDL group (P<0.01), which indicated that TCS could promote the formation of autophagy.Conclusion:TCS effectively reduced the serum lipid of atherosclerosis mice, significantly reduced relative aortic plaque area. In vitro, TCS significantly reduced phagocytosis of macrophages on lipid and formation rate of foam cells. In terms of mRNA, TCS down-regulated the mRNA expression of CD36 and SR-A1 while up-regulated mRNA exrepssion of ABCA1 and ABCG1. Further, it also increased the level of autophagy in the cells, and promoted the process of autophagy. |
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