养阴活血方干预ApoE-/-动脉粥样硬化小鼠DNA甲基化酶的研究  

Effects of Yangyin Huoxue Prescription on DNA Methylase in ApoE-/-Atherosclerosis Mice

作　　者：缪蓉 周李煜 白丹丹 杨云君 陈艳冉 姜林宏 袁冬平[1] 龙军[1] 吉娟 MIAO Rong;ZHOU Li-yu;BAI Dan-dan;YANG Yun-jun;CHEN Yan-ran;JIANG Lin-hong;YUAN Dong-ping;LONG Jun;JI Juan(School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China;Hospital for Skin Disease,Institute of Dermatology of Chinese Academy of Medical Sciences,Nanjing 210042,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]中国医学科学院皮肤病医院/中国医学科学院皮肤病研究所,江苏南京210042

出　　处：《南京中医药大学学报》2023年第6期523-531,共9页Journal of Nanjing University of Traditional Chinese Medicine

基　　金：国家自然科学基金面上项目(81973823,81573929);2021年江苏省研究生实践创新计划(SJCX210678)。

摘　　要：目的从DNA甲基化角度观察养阴活血方及其功效单元干预动脉粥样硬化(Atherosclerosis,AS)进程。方法ApoE-/-小鼠随机分为对照组、模型组、辛伐他汀组、养阴活血方组、养阴组、清热组和活血组。对照组小鼠给予普通饲料喂养,其余组小鼠均给予高脂饲料喂养。分别于第10、12、14、16、18周,对照组小鼠注射等体积无菌PBS,其余组腹腔注射10μg的LPS。灌胃给药与高脂饲料喂养同时进行,第20周检测。油红O染色观测小鼠主动脉斑块面积;生化试剂盒检测小鼠TC、TG、LDL-C、HDL-C、non-HDL-C水平;HE冠脉染色观测小鼠冠脉管腔狭窄情况和管腔面积;qPCR检测小鼠脾脏/主动脉DNA甲基化酶Tet1、Tet2、Tet3、Dnmt1、Dnmt3a、Dnmt3b mRNA表达;Western blot检测小鼠脾脏/主动脉DNA甲基化酶Dnmt1、Dnmt3a、Dnmt3b蛋白表达;ELISA检测细胞因子IL-6、IL-2、TGF-β1的变化。结果与模型组相比,养阴活血方组能显著减少斑块面积并抑制冠脉狭窄(P<0.01),降低TG、LDL-C水平(P<0.01),升高HDL-C水平(P<0.05),降低脾脏Dnmt1和Dnmt3a蛋白表达(P<0.05),降低主动脉Dnmt1 mRNA、Dnmt3b mRNA(P<0.05,P<0.001)和主动脉Dnmt1、Dnmt3b蛋白表达(P<0.05),下调血清IL-6水平(P<0.05),上调TGF-β1水平(P<0.05)。与模型组比较,各功效单元组均不同程度减少斑块面积、调节血脂水平;养阴组降低脾脏Dnmt3b mRNA表达(P<0.05),降低主动脉Dnmt3b mRNA(P<0.001)和Dnmt3a蛋白(P<0.05)表达,升高TGF-β1水平(P<0.05);清热组降低脾脏Dnmt1蛋白表达(P<0.05),主动脉Dnmt1和Dnmt3b mRNA(P<0.05)和Dnmt1、Dnmt3a、Dnmt3b蛋白表达(P<0.05),降低IL-6水平(P<0.05),增加TGF-β1水平(P<0.05);活血组降低主动脉Dnmt3a和Dnmt3b蛋白表达(P<0.05),降低IL-6水平(P<0.05)。结论养阴活血方通过调节血脂,降低DNA甲基化酶Dnmts的表达,改善炎症水平,从而有效减少高脂联合LPS注射诱发的ApoE-/-小鼠AS斑块的形成。OBJECTIVE To investigate the effect of Yangyin Huoxue Prescription and its functional units on the process of Atherosclerosis(AS)from the perspective of DNA methylation.METHODS ApoE-/-mice were randomly divided into the control group,model group,simvastatin group,Yangyin Huoxue Prescription group,Yangyin group,Qingre group and Huoxue group.The mice in the control group were fed with normal diet and the mice in the other groups were fed with high-fat diet.At the 10th,12th,14th,16th and 18th week,the control group was injected with an equal volume of sterile PBS,and the other groups were injected with 10μg LPS intraperitoneally.Intragastric administration was carried out simultaneously with high-fat diet feeding and ended at the 20th week when evaluations were carried out.The area of aortic plaque in mice was observed by oil red O staining;the levels of TC,TG,LDL-C,HDL-C and non-HDL-C in mice were detected by kits;HE staining was used to observe the stenosis and lumen area of coronary artery in mice;the mRNA expression of DNA methylation enzymes Tet1,Tet2,Tet3,Dnmt1,Dnmt3a and Dnmt3b in spleen/aorta of mice was detected by qPCR;western blot was used to detect the expression of Dnmt1,Dnmt3a and Dnmt3b proteins in the spleen/aorta of mice;ELISA was used to detect the changes of cytokines IL-6,IL-2 and TGF-β1.RESULTS Compared with the model group,Yangyin Huoxue Prescription could significantly reduce the plaque area and inhibit coronary stenosis(P<0.01),reduce the levels of TG and LDL-C(P<0.01),increase the level of HDL-C(P<0.05),reduce the expression of Dnmt1 and Dnmt3a proteins in spleen(P<0.05),reduce the expression of Dnmt1 mRNA and Dnmt3b mRNA in aorta(P<0.001,P<0.05)and the expression of Dnmt1 and Dnmt3b proteins in aorta(P<0.05).The level of serum IL-6 was down-regulated(P<0.05)and TGF-β1 was up-regulated(P<0.05).Compared with the model group,each efficacy unit group reduced plaque area and regulated blood lipid levels;the Yangyin group decreased the expression of Dnmt3b mRNA in spleen(P<0.05),reduced the ex

关 键 词：养阴活血方 动脉粥样硬化 APOE-/-小鼠 斑块 血脂 DNA甲基化 

分 类 号：R285.5[医药卫生—中药学]