| ObjectiveAcute cardio cerebrovascular events caused by atherosclerotic vascular diseases and rupture of AS plaques are the leading cause of death and disability in patients with cardiovascular and cerebrovascular diseases.The major pathological features of AS include chronic inflammation,oxidative stress and lipid accumulation in the walls of the large and middle arteries.Finding anti-inflammatory and antioxidant active substances is one of the important directions for the development of anti AS drugs.Indole-2,3-dione,also known as indole quinone,is widely distributed in the body and has many pharmacological activities such as anti-inflammatory,antioxidant and so on.It may be an ideal anti AS substance.This study was designed to investigate the effect of ISA on AS and plaque stability in AS animal models using ISA alone and a combination of classical lipid-lowering agents,and try to analyze possible mechanisms.Methods1.grouping and model establishment of experimental animalsIn this study,6-8 weeks old male apoE-/-mice were randomly divided into six groups: blank control group(1.25% of tragacanth),positive control group(simvastatin 5mg/kg),ISA low,medium and high dose(25mg/kg,50mg/kg,100mg/kg)and combined therapy group(ISA50mg/kg + simvastatin 5mg/kg).Each group of 12-13 mice were fed with high fat and high cholesterol diet(fat 15.8% and cholesterol 1.25%).The rats were given saline(1.25% tragacanth)or drug once a day.After 12 weeks,the mice were killed and the blood and tissue samples were taken.2.The Evaluation of body weight and general condition in miceThe body weight of the mice was weighed every week and the normal state(intragastri-cresistance,skin gloss and degree of neck swelling)were observed and recorded.3.Determination of blood lipid levelsAt the end of the fourth week,mice were sacrificed 50 ml after fasting,and plasma was separated.The total cholesterol(TC)and triglyceride(TG)contents were measured by enzymatic method.4.Determination of lipid oxidative indexAt the end of the 8th week,the mice were fasted and the plasma of the inner canthal was 50 ml.The plasma was separated and the contents of superoxide dismutase(SOD)and malondialdehyde(MDA)were measured by spectrophotometry.5.Determination of plasma inflammatory factorsThe levels of tumor necrosis factor-?(TNF-?)and interleukin-6(interleukin-6)were measured by enzyme-linked immunosorbent assay(ELISA).6.Histopathological examinationThe whole aorta was isolated from the mice,and the intima was exposed and exposed.The adipose tissue of the periosteum was discarded and stained with oil red O.The roots of the aorta were embedded and frozen sections were stained with oil red O stained AS Plaque,respectively,to calculate the area of aortic intimal plaque and aortic root patch area and analysis.7.plaque stability evaluationImmunohistochemical method was used to determine the content of macrophages(positive region of MOMA-2 antibody)and smooth muscle cells(positive region of ?-SM-actin antibody)in mouse aortic root plaque;lipid content was determined by oil red O and Masson content of collagen staining method.The stability of AS plaques was evaluated using the formula: plaque stability score =(smooth muscle cell area + collagen area)/(macrophage area + lipid area).8.statistical analysis methodThe experimental data were expressed in the form of x ±s;the independent sample t tests were conducted in two groups;the statistical results were analyzed using GraphPad Prism 6 software;the image information was analyzed by Image-Pro Plus 6 software.P<0.05 indicated that the difference was statistically significant.Results1.During the course of administration,there was no significant difference in weight between the experimental groups and the blank control group.The general state of the mice was evaluated at the end of the administration.The experimental groups were more active than the controls(increased by 34%,34%,44%,36% and 36%),indicating that ISA significantly improved the general state of mice.2.To determine the content of TC and TG in plasma by enzyme method: Compared with the blank control group,only TC content decreased in simvastatin group(simvastatin group VS.control group reduce 23%),the other groups had no significant difference;there was no significant difference in TG content.3.SOD activity and MDA content in plasma were detected by spectrophotometry.The SOD activity and MDA content in each experimental group were not significantly different from those in the blank control group.4.The content of TNF-? and IL-6 in plasma were measured by ELISA.The levels of TNF-? were significantly decreased in the experimental groups compared with the blank control group(decreased by 42%,23%,35%,19% and 26%);Compared ISA middle dose group with the blank control group,the content of IL-6 was significantly decreased(ISA middle dose group vs.blank control group reduce 38%).5.ISA can significantly reduce the aortic intimal plaque burden(decreased by 46%,42%,44%,34% and 36%)and the aortic root patch area(decreased by 51%,35%,36%,25% and 32%).6.ISA can significantly reduce the macrophage content of aortic root plaque(decreased by 72%,68%,70%,57% and 50%)and the lipid content(decreased by 34%,29%,36%,26% and 34%).ISA can significantly increased the content of smooth muscle cells(increased by 160%,29%,89%,155% and 87%).The content of collagen in the aortic root plaque was not significantly changed.The plaque stability score showed that ISA can significantly increase plaque stability(increased by 3.29 fold,1.09 fold,2.22 fold,1.81 fold and 1.87 fold).Conclusion1.ISA can reduce AS damage and significantly improve plaque stability.2.The anti-AS activity of ISA may be related to its anti-inflammatory mechanism,which is not related to blood lipid changes and oxidative stress. |
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