| Objective: Atherosclerosis （AS） was the major pathological basis ofsome cardiovascular and cerebrovascular events，that referred to disorder oflipid metabolism, endothelial dysfunction and the disorder of mechanism ofcruor. Therefore, according to the complicated pathologic process of AS thatcaused by multiple factors, a treatment proposal named “Golden Triangle”（ATS）, which consisted of Atorvastatin, Tongxinluo, and Aspirin wasproposed. In this study, high-fat diet induced ApoE-/-mice AS model wereused to investigate the role of the ATS project in the AS prevention, theadvantages and its mechanism of the three drugs combination in AS treatment.This study will provide more reasonable clinical prevention projects forprevention and cure ischemic cardiovascular and cerebrovascular disease.Methods:1The protective effects of ATS in ApoE-/-mouse fed with high-fat diet andthe change of vascular morphology and blood lipidsOne hundred and four ApoE-/-mice ingested high-fat diet were randomlydivided into seven groups （n=20）: vehicle group, Atorvastatin group （ATO）,Aspirin group （ASP）, Tongxinluo group （TXL）, ATO+ASP group （A+A）,ATS low-dose group （ATS-L）, high-dose group （ATS-H）. Some otherC57BL/6J mice were used for the normal control group, ingested the normaldiet. The mice were administered gavages for12weeks. The serum ofanimals was adopted to detect the content of TC、TG、HDL、LDL and COX-1.The pathological and ultrastructural changes in aortal tissue were determinedwith HE staining and transmission electron microscopy （TEM）. The changeof aortal atherosclerotic lesions and lipid deposition in atheromatous plaquewas determined with Oil Red O staining.2The protective effects of ATS on the endothelial dysfunction in ApoE-/- mouseExperimental modeling, grouping, administration and derived methodwere the same with method1. The serum was adopted to detect the content ofNO with Nitrale reduetase, the activity of SOD with hydroxylamine method,and the content of hs-CRP、ox-LDL with ELISA. Immunohistochemisty wasused to detect the expression of ET-1and Mac-3. Western-blot was used toanalyze the protein expression of COX-2、ICAM-l、VCAM-l、MCP-1、GM-CSF.3The effects of ATS on the NF-κB signaling pathway in aorta of ApoE-/-mouse caused by ASExperimental modeling, grouping, administration and derived methodwere the same with method1. Western blot was used to analyze the proteinexpression of IκKβ, IκBa, p-IκBa and NF-κBp65in aortal tissue. Real-timequantitative PCR was used to analyze the mRNA expression of IκKβ, IκBa,and NF-κB p65in aortal tissue.Results:1The protective effects of ATS in ApoE-/-mouse fed with high-fat diet andthe change of vascular morphology and blood lipids1.1The pathological and ultrastructural of aorta: In the vehicle group, plentyof foam cells accumulated in the subendothelial and the tunica media, thethickened vessel wall, atheromatous plaque were observed significantly withHE staining; The defluvium of endothelial cell, the vacuolization ofmitochondria and endoplasmic reticulum, the destruction of intercellularconnection, the broadening of subendothelial gap, a large number of lipiddroplet and cholesterol crystal in cytoplasm of smooth muscle were observedwith TEM. The injury of aorta tissue in medication administration groups wassignificantly reduced. Compared with the vehicle group and other medicationadministration groups, the aortal tissue injury of ATS-H group wassignificantly reduced.1.2The oil red O staining of aortal: In the vehicle group, a large number ofred plaque and red lipid droplet were observed, aortic was less flexible, and the plaque was easy to fall off. In the drug group, the number of red plaquewas reduced, the plaque was more stable, and the red lipid droplet wasreduced, especial in the ATS-H group.1.3The levels of lipids in serum: Compared with the normal group, thecontent of CHO, TG, LDL were significantly increased （P<0.01）, the contentof HDL was significantly decreased （P<0.01） in vehicle group. Comparedwith the vehicle group, the content of CHO,TG,LDL were significantlydecreased （P<0.01）, the content of HDL was significantly increased （P<0.01,P<0.05） in TXL and ATO group，but no significant difference between thetwo groups （P>0.05） was observed; in both ATS low and high dose groups,the content of CHO,TG,LDL were lower than that in the ASP, TXL and A+Agroup （P<0.05or P<0.01）, but the content of HDL were higher than that inthe ASP,TXL and A+A group （P<0.05or P<0.01）, in ATS-H group, thecontent of CHO,LDL were lower than that in the ATS-L group （P<0.05）, butthe content of HDL was higher than that in the ATS-L group （P<0.05）.1.4The content of COX-1in serum: Compared with the normal group, thecontent of COX-1was significantly increased （P<0.01） in vehicle group;Compared with the vehicle group, the content of COX-1was significantlydecreased （P<0.05,P<0.01） in TXL and ASP group, but no significantdifference was observed between the two groups（P>0.05）; in the ATS-L andATS-H groups, the content of COX-1were lower than that in the ASP,TXLand A+A group （P<0.05, P<0.01）, but no significant difference was observedbetween the two groups （P>0.05）.2The effects of ATS on the endothelial dysfunction in ApoE-/-mouse2.1The content of NO in serum: Compared with the normal group, thecontent of NO was significantly decreased （P<0.01） in vehicle group;Compared with the vehicle group, the content of NO were significantlyincreased （P<0.05, P<0.01） in TXL and ATO group, but no significantdifference was observed between the two groups （P>0.05）; The content of NOin the ATS-L and ATS-H groups were higher than that in the ASP, TXL andA+A group （P<0.05, P<0.01）, but no significant difference was observed between the two groups （P>0.05）.2.2The positive expression of ET-1in aortal tissue: In normal group, theweak positive staining of ET-1could be observed in aortal endothelial cellswith the immunohistochemisty. The positive staining could be observed invehicle group, the positive expression of ET-1was significantly decreasedat different extent in treatment group other than the ASP group, and in theATS-L and ATS-H groups,the positive expression of ET-1were significantlydecreased than that in the other groups.2.3The vigor of SOD and ox-LDL in serum: Compared with the normalgroup, the vigor of SOD in vehicle group was significantly decreased（P<0.01）, and the vigor of ox-LDL were significantly increased （P<0.01）.Compared with vehicle group, the vigor of SOD were significantly increased（P<0.01）, and the vigor of ox-LDL were significantly decreased （P＜0.05,P<0.01） in TXL and ATO group, but no significant difference was observedbetween the two groups （P>0.05）; The effect of SOD and ox-LDL in theATS-L and ATS-H groups were superior to that in ATO,TXL,A+A groups（P<0.05, P<0.01）, but no statistically difference was observed between thetwo groups （P>0.05）.2.4The content of hs-CRP in serum: Compared with the normal group, thecontent of hs-CRP in vehicle group was significantly increased （P<0.01）;Compared with the vehicle group, the content of hs-CRP in TXL,ATO andASP groups was significantly decreased （P<0.05, P<0.01）, but no significantdifference was observed among the three groups （P>0.05）; In ATS-L and A+Agroup, the content of hs-CRP was significantly decreased than that in theTXL,ATO and ASP groups （P<0.05）, but was significantly increased than thatin the ATS-H group（P＜0.05）.2.5The positive expression of Mac-3in aortal tissue: The plaque and thepositive expression of Mac-3were not observed in normal group; The plaqueand the positive expression of Mac-3were observed in vehicle group, and thepositive cells of Mac-3were deeply stained. The scope of plaque and thepositive expression of Mac-3were decreased at different extent in treatment groups, especially, the number of positive cells of Mac-3in ATS-Land ATS-Hgroups were significantly decreased than that in other groups.2.6The protein expression of inflammatory factors in aortal tissue: Comparedwith the normal group, the protein expression of ICAM-1, VCAM-1,MCP-1,COX-2and GM-CSF in aortal tissue were significantly increasedin the vehicle group （P<0.01）; Compared with the vehicle group, the proteinexpression of these inflammatory factors were significantly decreased in theTXL,ATO and ASP group （P<0.05or P<0.01）, but no significant differenceamong the three groups （P>0.05）; In the ATS-L and A+A group, the proteinexpression of these inflammatory factors were significantly decreased thanthat in the TXL, ATO and ASP groups （P<0.01,P<0.05）, but was significantlyincreased than that in the ATS-H group （P＜0.05）.3The effects of ATS on the NF-κB signaling pathway in the aortal tissue ofAS ApoE-/-mouse3.1The mRNA expression of IκKβ, IκBα and NF-κBp65in the aortal tissue:Compared with the normal group, the mRNA expression of IκKβ andNF-κBp65in the aortal tissue were significantly increased in the vehiclegroup （P<0.01）, but the mRNA expression of IκBα was significantlydecreased （P<0.01）. Compared with the vehicle group, in treatment groups,the mRNA expression of IκKβ, NF-κBp65were significantly decreased（P<0.05or P<0.01）, the mRNA expression of IκBα was significantlyincreased （P<0.05or P<0.01）, but no significant change of the mRNAexpression of IκKβ was observed in the ATO group. The mRNA expression ofIκKβ, NF-κBp65in ATS-H group were significantly decreased than that inA+A and ATS-L group （P<0.01, P<0.05）, but the mRNA expression of IκBαwas significantly increased than that in the A+A and ATS-L group （P<0.01, P<0.05）.3.2The protein expression of IκKβ, IκBα, p-IκBa and NF-κBp65in the aortaltissue: Compared with the normal group, the protein expression of IκKβ,p-IκBa and NF-κBp65were significantly increased in the vehicle group（P<0.01）, but the protein expression of IκBα was significantly decreased （P<0.01）.Compared with the vehicle group, in treatment groups, the proteinexpression of IκKβ, p-IκBa, NF-κBp65were significantly decreased （P<0.05or P<0.01）, the protein expression of IκBα was significantly increased（P<0.05or P<0.01）,but no significant change of the protein expression ofIκKβ was observed in the ATO group. The protein expression of IκKβ, p-IκBa,NF-κBp65in ATS-H group were significantly decreased than that in A+A andATS-L group （P<0.01, P<0.05）, but the protein expression of IκBα wassignificantly increased than that in the A+A and ATS-L group （P<0.01, P<0.05）.Conclusion:1The ATS project can enhance the effect of regulating the lipidmetabolism and inhibiting the aggregation of platelet. The protective effect ofthe ATS project was superior to the TXL, ATO, ASP used alone and thecombination of ATO and ASP. It played an important role from multipleperspectives in the treatment of AS;2The ATS project could significantly improve the endothelial function.The protective effect of the ATS project was superior to the TXL, ATO, ASPused alone and the combination of ATO and ASP. The mechanism may berelated to inhibit the lipid peroxidation and inflammatory reaction;3The ATS project could inhibit the inflammatory reaction throughinhibiting the vigor of IκKβ, reducing the phosphorylation of IκBα and thedegradation of IκBα;The ATS project could protect the vascular endothelium and delay theprogression of AS through regulating lipid metabolism, inhibiting bloodcoagulation, inhibiting oxidative damage, and inhibiting vascularinflammatory reaction according to disturb the IκKα/IκBβ/NF-κB signalingpathway. At the same time, the ATS project could reduce the dose of ATO andASP. |
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