养阴活血方干预ApoE<sup>-/-</sup>动脉粥样硬化小鼠DNA甲基化酶的研究

缪蓉; 周李煜; 白丹丹; 杨云君; 陈艳冉; 姜林宏; 袁冬平; 龙军; 吉娟

doi:10.14148/j.issn.1672-0482.2023.0523

养阴活血方干预ApoE^-/-动脉粥样硬化小鼠DNA甲基化酶的研究

doi: 10.14148/j.issn.1672-0482.2023.0523

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

基金项目:

国家自然科学基金面上项目 81973823

国家自然科学基金面上项目 81573929

2021年江苏省研究生实践创新计划 SJCX210678

详细信息

作者简介:
缪蓉, 女, 硕士研究生, E-mail: mr971229@163.com

通讯作者:
龙军, 男, 副教授, 主要从事心血管药理学研究, E-mail: longjun@njucm.edu.cn

中图分类号: R285.5
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- 被引次数: 0
出版历程
- 收稿日期: 2022-12-01
- 网络出版日期: 2023-06-12
- 发布日期: 2023-06-10

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

1.
School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2.
Hospital for Skin Disease, Institute of Dermatology of Chinese Academy of Medical Sciences, Nanjing 210042, China

摘要

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10 μg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。
- 养阴活血方 /
- 动脉粥样硬化 /
- ApoE^-/-小鼠 /
- 斑块 /
- 血脂 /
- DNA甲基化
Abstract: 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 10 μg 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 expression of Dnmt3b mRNA (P < 0.001) and Dnmt3a protein in aorta (P < 0.05), and increased TGF-β1 (P < 0.05). The Qingre group decreased the expression of Dnmt1 protein in spleen (P < 0.05), Dnmt1 and Dnmt3b mRNA in aorta (P < 0.05) and the expression of Dnmt1, Dnmt3a and Dnmt3b proteins (P < 0.05), reduced the level of IL-6 (P < 0.05) and increased the level of TGF-β1 (P < 0.05). In the Huoxue group, the expression of Dnmt3a and Dnmt3b protein in aorta was decreased (P < 0.05), and the level of IL-6 was decreased (P < 0.05). CONCLUSION Yangyin Huoxue Prescription can effectively reduce the formation of AS plaques in ApoE^-/-mice induced by high fat combined with LPS injection by regulating blood lipids, reducing the expression of DNA methylase Dnmts and improving the level of inflammation.
- Yangyin Huoxue Prescription /
- atherosclerosis /
- ApoE^-/- mice /
- plaque /
- lipids /
- DNA methylation

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图 1 各组主动脉斑块油红O染色(n=3)和冠脉HE染色(n=5)

注: A.油红O染色；B.HE染色；a.对照组; b.模型组; c.辛伐他汀组; d.养阴活血方组; e.养阴组; f.清热组; g.活血组。与对照组比较, ^#P < 0.05, ^##P < 0.01;与模型组比较, ^*P < 0.05, ^**P < 0.01。x±s。

Figure 1. Aortic plaque oil red O staining (n=3) and coronary artery HE staining(n=5)

下载: 全尺寸图片幻灯片

图 2 养阴活血方及其功效单元对ApoE^-/-小鼠体质量及血脂水平的影响

注: 与对照组比较, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001;与模型组比较, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001。x±s，n=6。

Figure 2. Effects of Yangyin Huoxue Prescription and its efficacy units on weight and serum lipid levels of ApoE^-/- mice

下载: 全尺寸图片幻灯片

图 3 养阴活血方及其功效单元对ApoE^-/-小鼠脾脏和主动脉Tets、Dnmts mRNA表达的影响

注: 与对照组比较, ^#P < 0.05, ^###P < 0.001;与模型组比较, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001。x±s，n=3。

Figure 3. Effects of Yangyin Huoxue Prescription and its efficacy units on mRNA expression of Tets and Dnmts in spleen and aorta of ApoE^-/-mice

下载: 全尺寸图片幻灯片

图 4 养阴活血方及其功效单元对ApoE^-/-小鼠脾脏和主动脉Dnmt1、Dnmt3a、Dnmt3b蛋白表达的影响

注: 与对照组比较, ^#P < 0.05, ^##P < 0.01;与模型组比较, ^*P < 0.05, ^**P < 0.01。x±s，n=3。

Figure 4. Effects of Yangyin Huoxue Prescription and its efficacy units on protein expression of Dnmt1, Dnmt3a, Dnmt3b in spleen and aorta of ApoE^-/- mice

下载: 全尺寸图片幻灯片

图 5 养阴活血方及其功效单元对ApoE^-/-小鼠细胞因子的影响

注: 与对照组比较, ^#P < 0.05, ^##P < 0.01;与模型组比较, ^*P < 0.05。x±s，n=5。

Figure 5. Effects of Yangyin Huoxue Prescription and its efficacy units on cytokines of ApoE^-/- mice

下载: 全尺寸图片幻灯片

表 1 qPCR引物序列

Table 1. Primer sequences used in qPCR

mRNA	上游引物序列(5′→3′)	下游引物序列(5′→3′)
Tet1	ACACAGTGGTGCTAATGCAG	AGCATGAACGGGAGAATCGG
Tet2	AGAGAAGACAATCGAGAAGTCGG	CCTTCCGTACTCCCAAACTCAT
Tet3	TGCGATTGTGTCGAACAAATAGT	TCCATACCGATCCTCCATGAG
Dnmt1	ATCCTGTGAAAGAGAACCCTGT	CCGATGCGATAGGGCTCTG
Dnmt3a	GAGGGAACTGAGACCCCAC	CTGGAAGGTGAGTCTTGGCA
Dnmt3b	AGCGGGTATGAGGAGTGCAT	GGGAGCATCCTTCGTGTCTG
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT

下载: 导出CSV

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