Mechanism Study Of Danloutablets Promoting Re-endothelialization After Drug Eluting Stent Implantation On Rabbit Abdominal Aorta Atherosclerosis Model

ObjectiveTo study the effect and possible mechanism of Danloutablets promoting re-endothelialization after drug eluting stent implantation on rabbit abdominal aorta atherosclerosis model.Method1 Preparation of rabbit abdominal aorta atherosclerosis model and group dividing: New Zealand Male rabbits(clean grade)were fed with high-fat diet,and received abdominal aorta balloon injury 1 week after,and high-fat feeding for 7 weeks then,stents would be implanted in the position of medicine stenosis showed by abdominal aorta angiography,then all rabbits were randomly divided into 4 groups,that is,the control group(isotonic saline),rosuvastatin group(1mg/kg · d),Danloutablets group(0.5g/kg · d),Danloutablets combined with rosuvastatin group(Danloutablets 0.5g/kg·d+ rosuvastatin 1mg/kg·d),with 7 rabbits in each group.Each group will be intervented by corresponding drug after the model finished.through the method of gavage administration for 12 weeks.2 Blood lipids,liver and kidney function detection:Blood lipids,liver and kidney function(TC,TG,ALT,AST,Cr)was checked by automatic biochemical analyzer before and after PCI operation for 12 weeks in each group.3 The serum levels of CRP,MCP-1,VEGF,MMP-9 and SDF-1:CRP,MCP-1,VEGF,MMP-9,and SDF-1 levels in serum of each group were detected by the method of ELISA after drug intervention for 12 weeks.4 The result of stent slice HE staining after drug intervention for 12 weeks.5 The expression of CD34 and vWF:The expression of CD34 and vWF in each group were detected by immunohistochemical staining.6 Evaluation of stent strut coverage: Through OCT imaging,percent stent struts coverage,lumen area,neointimal thickness,neointimal area were dected.7 Re-endothelialization:Endothelial cell crawling,neointimal coverage,platelet adhesion and aggregation were evaluated by scanning electron microscopy.8 The detection of related signal transduction protein:The expression of p-PI3 K,p-Akt and eNOS in the adjacent segments of the abdominal aorta were measured by Western-Blot assay after drug intervention.Result1 Animal model:After balloon injuring and high fat feeding for 8 weeks,angiography of abdominal aorta showed severe stenosis(30~90%).Pathological staining: Normal rabbits abdominal aorta HE staining showed that aortic intima was smooth and intact with no lipid and cholesterol crystal deposition.After abdominal aorta balloon injury and high fat diet for 12 weeks,abdominal aortic HE staining showed intimal thickening,cholesterol crystal deposition with lumen eccentric stenosis,structure morphology changes were also visible in renal and liver tissue because of lipid deposition.The drug eluting stent was implanted on the basis of atherosclerotic stenosis.And vessel angiography showed patency without poor adherence to the stent.2 Blood lipids,liver and kidney function:Automatic biochemical analyzer showed no significant difference(P>0.05)before PCI operation intervention in TG,TC and LDL-c levels of each group.Serum lipid levels reduced in Danloutablets group,rosuvastatin group and mixed group(P<0.05)group when compared with before PCI operation,and the drop of rosuvastatin group is more than Danloutablets group(P<0.05),and the drop of mixed group was higher than that of rosuvastatin group(P<0.05).There was no significant difference of renal and liver function detection before and after PCI operation(P>0.05).3 Serum inflammatory factors: ELISA assay suggested that serum CRP and MCP-1 levels of control group was higher than other groups,and the drop of mixed group is higher than rosuvastatin group and Danloutablets group,the difference was statistically significant(P<0.05).Serum VEGF,MMP9,SDF-1,eNOS levels of mixed group,rosuvastatin group and Danloutablets group was higher than control group,and rosuvastatin was higher than that of Danloutablets group and mixed group was higher than that of rosuvastatin group(P<0.05).4 OCT imaging:OCT imaging of abdominal aorta showed that stents were covered with intact intima,and there was no significant difference in the coverage rate of stent struts(P>0.05).NIH Area,neointimal thickness(NIH Thickness)in control group was significantly higher than that of other groups(P<0.05).Luman Area of control group was significantly lower than that of the other groups,and the effect of rosuvastatin group to reduce the thickness and area of neointima was stronger than that of Danloutablets group,mixed group was stronger than that of rosuvastatin group(P<0.05)5 The pathological change of stent segment vascular tissue:HE staining showed that the stent surface was covered by intact endothelial cells.Intimal hyperplasia with formation of in-stent atherosclerosis was observed in control group,while the intimal of other groups were smooth and intact with no stenosis.6 Immunohistochemical staining of stent segment vessels:Immunohistochemical staining indicated that the expression of CD34 and vWF(neointimal endothelial cell surface marker)were not sufficient in control group,and the expression of these markers increased in Danloutablets group and rosuvastatin group,especially showed mixed group of in membrane surface expression,suggesting showed well proportioned the mixed group.7 SEM scanning: TESCAN scanning electron microscopy showed that all stent specimens were covered by endothelial cells.The number of endothelial cells in control group were less than other groups.Even though the cell numbers of rosuvastatin group was more than Danloutablets group in high magnification,the cell morphology of latter group was more intact than former group.The endothelial cells of mixed group were arranged closely and more intact to normal morphology.8 The expression of related signal transduction protein: Western-Blot essay showed that the expression of p-PI3 K,p-Akt and eNOS protein in Danloutablets group,rosuvastatin group and mixed group were significantly higher than control group,(P<0.05),and rosuvastatin group was higher than that of Danloutablets group and mixed group was higher than rosuvastatin group(all P<0.05).Conclusion1 Danloutablets have effects of reducing blood lipid,inhibiting inflammatory reaction,and achieving a complete re-endothelialization in rabbit abdominal aorta atherosclerosis model.The effects enhanced when combined with rosuvastatin.2 The effect of Danloutablets promting a complete re-endothelialization might be achieved through the regulation of PI3K/AKT signal transduction protein,and associated with increacing the level of p-PI3 K and p-AKT protein expression.