循环内皮微粒相关microRNA在巨噬细胞炎性反应诱发动脉粥样硬化发生中的作用机制

doi:10.3877/cma.j.issn.2095-1221.2018.06.005

目的

研究循环内皮微粒相关microRNA在巨噬细胞炎性反应诱发动脉粥样硬化（AS）发生的作用机制。

方法

选取实验组和对照组SPF级5周小鼠各50只。实验小鼠分4组：对照组、动脉粥样硬化模型组（AS模型组）、NC-miRNA组和miRNA-19b抑制剂组，采用油红染色对AS病变的组织学进行观察，计算斑块面积相对比例，并检测生化指标的表达水平，主要包括血清中高密度脂蛋白胆固醇（HDL-C）、胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）和甘油三酯（TG），ELISA检测血管炎症因子TNF-α、IL-1、IL-6和IL-10的变化，Western Blot检测凋亡相关蛋白Bcl-2、cleaved-caspase-3和Bax表达的变化，实时荧光定量PCR检测miR-19b表达，流式细胞法检测巨噬细胞的凋亡率。多组间比较采用单因素方差分析或者重复测量的方差分析，两两比较采用LSD-t检验。

结果

各组胸腔动脉斑块面积百分比：对照组为（0.00±0.00）%；AS模型组为（9.59±6.53）%；NC-miRNA组为（8.96±3.47）%；miRNA-19b抑制剂组为（3.21±2.03）%，三组间比较差异有统计学意义（F = 20.572，P = 0.002）。与对照组比较，AS模型组和NC-miRNA组TC[AS模型组（3.26±0.21）mmol/L；NC-miRNA组（3.13± 0.14）mmol/ L；F = 13.994，P = 0.002]、TG[AS模型组（0.25±0.06）mmol/L；NC- miRNA（0.21±0.02）mmol/L；F = 11.230，P = 0.011）]和LDL-C [AS模型组（1.65±0.11）mmol/ L；NC-miRNA （1.59± 0.27）mmol/L；F = 10.069，P = 0.006）]水平升高，而HDL-CAS模型组[（0.08±0.09）mmol/L；NC-miRNA （0.08±0.05）mmol/L；F = 12.450，P = 0.004）]水平含量降低。与模型组比较，miRNA-19b抑制剂组TC（1.85±0.06）mmol/L、TG（0.15±0.03）mmol/ L和LDL-C（1.21±0.10）mmol/L水平降低，HDL-C （0.11±0.05）mmol/L水平则升高（P均< 0.05）。AS模型组与对照组相比，IL-1（34.06±3.58）g/L、IL-6（92.57±31.97）g/L和TNF-α（63.01±15.65）g/L水平升高，而IL-10（16.86±1.29）g/L的水平降低，NC-miRNA组各指标亦有相同的变化趋势；miRNA-19b抑制剂组IL-1（24.85±6.21）g/L、IL-6（53.29±17.15）g/ L和TNF-α（34.51±6.47 ）g/L水平低于AS模型组和NC-miRNA组（F = 13.671，P = 0.002），IL-10（24.94±4.72）g/L的水平则高于两组（F = 13.675，P = 0.008）。和对照组相比，AS模型组和NC- miRNA组的Bax（AS模型组3.39±0.01；NC-miRNA组0.64±0.02；F = 26.910，P = 0.000）和cleaved-PARP （AS模型组2.47±0.05；NC- miRNA组3.27±0.01；F = 13.226，P = 0.000）表达量升高。但Bcl-2（AS模型组0.67±0.02；NC-miRNA组0.64±0.02；F = 12.585，P = 0.000）表达水平下调。与NC- miRNA组相比，miRNA-19b抑制剂组的促细胞凋亡的蛋白Bax（2.16±0.02）和cleaved-PARP（1.91±0.04）表达水平下调，Bcl-2（1.05±0.01）的表达水平上调。与对照组比较，AS模型组（2.71±0.02）和NC-miRNA组（2.43±0.02）的miRNA- 19b的转录水平增加，差异有统计学意义（P均< 0.05），miRNA-19b抑制剂组（1.52±0.01）转录水平有升高，但相较于AS模型组和NC-miRNA组的变化其增加量不显著，（F = 15.353，P = 0.002）。miRNA-19b基因转录水平：对照组为1.02±0.03；AS模型组为2.71±0.02；NC- miRNA组为2.43±0.02；miRNA- 19b抑制剂组为1.52±0.01（P均 < 0.05）。细胞凋亡率：对照组为（4.41±0.18）%；AS模型组为（7.16±0.73）%；NC-miRNA组为（6.29±0.24）%；miRNA-19b抑制剂组为（5.01±0.11）%，（F = 12.889，P = 0.008）。

结论

miRNA-19b抑制剂可以减少巨噬细胞的凋亡，降低促炎症因子，可减少由于AS引起的血管厚度的增加，减少AS斑块的面积比例，使血脂中的TC、TG和LDL-C水平降低，HDL-C水平则有升高，这对冠心病的临床诊疗具有重要的临床指导意义。

关键词: 内皮微粒, miRNA-19b, 巨噬细胞, 炎症因子, 动脉粥样硬化

Objective

To study the circulating endothelial microparticles related microRNA in the atherosclerosis induced by inflammatory response of macrophage.

Methods

Five-week-old mice of SPF-grade were randomized to an experimental group and a control group (n = 50 for each group). The experimental mice were divided into four groups: a control group, an atherosclerosis (AS) model group, an NC-microRNA group and a microRNA-19 b inhibitor group. The histology of atherosclerotic lesions was observed with oil red staining, and the plaque area ratio was calculated. Serum cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C), TNF-α, IL-1, IL-6 and IL-10 were measured. The expressions of apoptosis related proteins Bcl-2, cleaved-caspase-3 and Bax were detected with Western Blot, the mir-19b expression was detected with real-time quantitative PCR, and the apoptosis rate of macrophages was detected with flow cytometry. Then the comparison among groups were analyzed with variance by single-factor or repeated measures, the comparison between two groups were analyzed with LSD-t test.

Results

The percentage of plaque area of thoracic artery in each group: (0.00±0.00)% in the control group, (9.59±6.53)% in the AS model group, (8.96±3.47)%in the NC-microRNA group and (3.21±2.03)% in the microRNA19b inhibitor group, and the differences of the three group were statistically significant (F = 20.572, P = 0.002). Compared with the control group, TC (TC) in the AS model group and the NC-microRNA group were as follows: TC[AS model group (3.26±0.21) mmol/L; NC-microRNA group (3.13±0.14) mmol/ L; F = 13.994, P = 13.994, P = 0.002], TG [AS model group 0.25 (0.25±0.06) mmol/ L; AS model group (1.65±0.11) mmol/L; NC-miRNA (1.59±0.27) mmol/L; F = 10.069, P = 0.006) increased, and HDL-C (AS) increased. In the model group, the levels of NC-microRNA (0.08±0.09) mmol/ L, NC-microRNA (0.08±0.05) mmol/L and F = 12.450, P = 0.004 decreased. Compared with the model group, the levels of TC (1.85±0.06) mmol/L, TG (0.15±0.03) mmol/ L and LDL-C (1.21±0.10) mmol/L decreased, while HDL-C (0.11±0.05) mmol/L increased in the miRNA- 19b inhibitor group (P < 0.05). Compared with the control group, the levels of IL-1 (34.06±3.58) g/ L, IL-6 (92.57±31.97) g/Land TNF-alpha (63.01±15.65)g/L increased, while the levels of IL-10 (16.86±1.29) g/L decreased, and the indexes of NC-microNA group also showed a same change trend; the levels of IL-1(24.85±6.21) g/L, IL-6 (53.29±17.15) g/L and TNF-alpha (34.51±6.47) g/ L in the inhibitor group of microRNA-19b were lower than those in the control group. The levels of IL- 10 (24.94±4.72 )g/L in Group A and NC-microRNA (F = 13.671, P = 0.002) were higher than those in Group B (F = 13.675, P = 0.008). Compared with the control group, Bax (AS model group 3.39±0.01; AS model group 3.39±0.01; NC- microRNA group 0.64 ±0.02; F = 26.910, P = 0.000) and cleaved-PARP (AS model group 2.47±0.05; NANC-microRNA group 3.27±0.01; F = 13.226, P = 0.000) expression increased. However, Bcl-2 (model group 0.67±0.02; NC-microRNA group 0.64±0.02; F = 12.585, P = 0.000) expression level was down-regulated. Compared with the NC-microRNA group, the expression levels of Bax (2.16±0.02) and cleaved-PARP (1.91±0.04) were down-regulated, while the expression levels of Bcl-2 (1.05±0.01) were up-regulated. Compared with the control group, the transcription level of microRNA-19b in the AS model group (2.71±0.02) and NC-microRNA group (2.43±0.02) D increased significantly (P < 0.05). The transcription level of the microRNA-19b inhibitor group (1.52±0.01) increased, but the increase was not significant (F = 15.353, P = 0.002) compared with the AS model group and NC-microRNA group. The transcription level of microRNA-19b gene was 1.02±0.03 in the control group, 2.71±0.02 in the AS model group, 2.43±0.02 in the NC-microRNA group and 1.52±0.01 in the microRNA-19b inhibitor group (P < 0.05). Apoptotic rate: (4.41±0.18)% in thecontrol group, (7.16±0.73)% in the AS model group, (6.29±0.24)% in the NC-microRNA group and (5.01±0.11)% in the microRNA19b inhibitor group, (F = 12.889, P = 0.008).

Conclusion

The inhibitor microRNA-19b can reduce macrophage apoptosis, decrease the pro-inflammatory factors, and reduce the development of blood vessel thickness caused by atherosclerosisi. It also can decrease the lipid TC, TG and LDL-C and increase the HDL-C, so that it may have active guiding significance for the clinical diagnosis and treatment of coronary heart disease.

Key words: Endothelial particles, Micrornas-19b, Macrophages, Inflammat-ory factors, Atherosclerosisi

表1 血脂相关性指标的水平测定结果（mmol/L，±s）

分组	动物数（只）	TC	TG	HDL-C	LDL-C
对照组	50	0.79±0.16	0.09±0.02	0.31±0.08	0.35±0.23
AS模型组	50	3.26±0.21^a	0.25±0.06^a	0.08±0.09^a	1.65±0.11^a
NC-miRNA组	50	3.13±0.14^ab	0.21±0.02^ab	0.08±0.05^ab	1.59±0.27^ab
miRNA-19b抑制剂组	50	1.85±0.06^ab	0.15±0.03^ab	0.11±0.05^ab	1.21±0.10^ab
F值		13.994	11.230	10.069	12.450
P值		0.002	0.011	0.006	0.004

表1 血脂相关性指标的水平测定结果（mmol/L，±s）

分组	动物数（只）	TC	TG	HDL-C	LDL-C
对照组	50	0.79±0.16	0.09±0.02	0.31±0.08	0.35±0.23
AS模型组	50	3.26±0.21^a	0.25±0.06^a	0.08±0.09^a	1.65±0.11^a
NC-miRNA组	50	3.13±0.14^ab	0.21±0.02^ab	0.08±0.05^ab	1.59±0.27^ab
miRNA-19b抑制剂组	50	1.85±0.06^ab	0.15±0.03^ab	0.11±0.05^ab	1.21±0.10^ab
F值		13.994	11.230	10.069	12.450
P值		0.002	0.011	0.006	0.004

图1 倒置光学显微下观察动脉血管组织病理变化（Oil-red染色，×600）

表2 各组小鼠血清IL-1、IL-6 IL-10和TNF-α含量比较（g/L，±s）

分组	动物数（只）	IL-1	IL-6	IL-10	TNF-α
对照组	50	23.51±5.01	45.23± 7.36	25.64±5.33	32.29± 5.39
AS模型组	50	34.06±3.58^a	92.57±31.97^a	16.86±1.29^a	63.01±15.65^a
NC-miRNA组	50	33.56±4.59^ab	89.26±21.07^ab	17.34±1.53^ab	60.91± 7.61^ab
miRNA-19b抑制剂组	50	24.85±6.21^ab	53.29±17.15^ab	24.94±4.72^ab	34.51± 6.47^ab
F值		13.671	17.592	13.675	20.362
P值		0.002	0.005	0.008	0.000

表2 各组小鼠血清IL-1、IL-6 IL-10和TNF-α含量比较（g/L，±s）

分组	动物数（只）	IL-1	IL-6	IL-10	TNF-α
对照组	50	23.51±5.01	45.23± 7.36	25.64±5.33	32.29± 5.39
AS模型组	50	34.06±3.58^a	92.57±31.97^a	16.86±1.29^a	63.01±15.65^a
NC-miRNA组	50	33.56±4.59^ab	89.26±21.07^ab	17.34±1.53^ab	60.91± 7.61^ab
miRNA-19b抑制剂组	50	24.85±6.21^ab	53.29±17.15^ab	24.94±4.72^ab	34.51± 6.47^ab
F值		13.671	17.592	13.675	20.362
P值		0.002	0.005	0.008	0.000

表3 BCL-2、cleaved-PARP以及Bax的蛋白相对表达量（±s）

分组	动物数（只）	BCL-2	cleaved-PARP	Bax
对照组	50	1.21±0.03	1.35±0.05	1.04±0.01
AS模型组	50	0.67±0.02^a	2.47±0.05^a	3.39±0.01^a
NC-miRNA组	50	0.64±0.02^ab	2.53±0.04^ab	3.27±0.01^ab
miRNA-19b抑制剂组	50	1.05±0.01^ab	1.91±0.04^ab	2.16±0.02^ab
F值		12.585	13.226	26.910
P值		0.013	0.009	0.000

表3 BCL-2、cleaved-PARP以及Bax的蛋白相对表达量（±s）

分组	动物数（只）	BCL-2	cleaved-PARP	Bax
对照组	50	1.21±0.03	1.35±0.05	1.04±0.01
AS模型组	50	0.67±0.02^a	2.47±0.05^a	3.39±0.01^a
NC-miRNA组	50	0.64±0.02^ab	2.53±0.04^ab	3.27±0.01^ab
miRNA-19b抑制剂组	50	1.05±0.01^ab	1.91±0.04^ab	2.16±0.02^ab
F值		12.585	13.226	26.910
P值		0.013	0.009	0.000

图2 Western Blot检测BCL-2、cleaved-PARP以及Bax表达

图3 miRNA-19b基因转录水平

1	Chrysohoou C, Kollia N, Tousoulis D. The Link between depression and atherosclerosis through the pathways of inflammation and endothelium dysfunction[J]. Maturitas, 2018, 109(5):1-5.
2	Jung YY, Kim KC, Park MH, et al. Atherosclerosis is exacerbated by chitinase-3-like-1 in amyloid precursor protein transgenic mice[J]. Theranostics, 2018, 8(3):749-766.
3	Katakami N. Mechanism of development of atherosclerosis and cardiovascular disease in diabetes mellitus[J]. J Atheroscler Thromb, 2018, 25(1):27-39.
4	Bornfeldt KE, Kramer F, Batorsky A, et al. A novel type 2 diabetes mouse model of combined diabetic kidney disease and atherosclerosis[J]. Am J Pathol, 2018, 188(2):343-346.
5	He PP, Jiang T, Ouyang XP, et al. Lipoprotein lipase: Biosynthesis, regulatory factors, and its role in atherosclerosis and other diseases[J]. Clinica Chimica Acta, 2018, 480(3):126-137.
6	Cochain C, Vafadarnejad E, Arampatzi PA, et al. Single-Cell RNA-Seq reveals the transcriptional landscape and heterogeneity of aortic macrophages in murine atherosclerosis[J]. Circ Res, 2018, 122(12):1661-1674.
7	Richardson G, Sage A, Bennaceur K, et al. Telomerase mediates lymphocyte proliferation but not the Atherosclerosis-Suppressive potential of regulatory T-Cells[J]. Arterioscler Thromb Vasc Biol, 2018, 38(6):1283-1296.
8	Zhong X, Ma X, Zhang L, et al. MIAT promotes proliferation and hinders apoptosis by modulating miR-181b/STAT3 axis in ox-LDL-induced atherosclerosis cell models[J]. Biomed Pharmacother, 2018, 97(1):1078-1085.
9	Zernecke A, Haring B, Selvin E, et al. Dietary protein sources and risk for incident chronic kidney disease:results from the atherosclerosis risk in communities(ARIC)study[J]. J Ren Nutr, 2018, 27(4):233-242.
10	Kim HJ, Cha GS, Kim HJ, et al. Porphyromonas gingivalis accelerates atherosclerosis through oxidation of high-density lipoprotein[J]. J Periodontal Implant Sci, 2018, 48(1):60-68.
11	Li CL, Li SF, Zhang F, et al. Endothelial microparticles-mediated transfer of microRNA-19b promotes atherosclerosis via activating perivascular adipose tissue inflammation in apoE(-/-) mice[J]. Biochem Biophys Res Commun, 2018, 495(2):1922-1929.
12	Huang Y, Qi Y, Du JQ, et al. Protosappanin a protects against atherosclerosis via anti-hyperlipidemia, anti-inflammation and NF-kappa B signaling pathway in hyperlipidemic rabbits[J]. Iran J Basic Med Sci, 2018, 21(1):33-38.
13	Qamar T, Norby FL, Aguilar D, et al. Galectin-3 and incidence of atrial fibrillation:The Atherosclerosis Risk in Communities(ARIC)study[J]. Am Heart J, 2018, 192(11):19-25.
14	Souilhol C, Goodspeed L, Wang S, et al. Deficiency of invariant natural killer T cells does not protect against obesity but exacerbates atherosclerosis in Ldlr-/- Mice[J]. Int J Mol Sci, 2018, 19(2): pii: E510.
15	Pirillo A, Xu D, Wang L. miR-26a inhibits atherosclerosis progression by targeting TRPC3[J]. Cell Biosci, 2018, 8(1):4-4.
16	Naugler C, Harmsen MC, Evans PC, et al. Endothelial-mesenchymal transition in atherosclerosis[J]. Cardiovasc Res, 2018, 114(4):565-577.
17	Tabib A, Leroux C, Mornex JF, et al. Accelerated coronary atherosclerosis and arteriosclerosis in young human-immunodeficiency-virus-positive patients[J]. Coron Artery Dis, 2000, 11(1):41-46.
18	Canakinumab A, Macfadyen JG, Thuren T, et al. Effect of interleukin-1β inhibition with canakinumab on incident lung cancer in patients with atherosclerosis:exploratory results from a randomised, double-blind,placebo-controlled trial[J]. Lancet, 2017, 390(10105):1833-1837.
19	Yuan N, Zhang HF, Wei Q, et al. Expression of CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells, interleukin 10 and transforming growth factor β in newly diagnosed type 2 diabetic patients[J]. Exp Clin Endocrinol Diabetes, 2018, 126(2):96-101.
20	Souilhol C, Harmsen MC, Evans PC, et al. Endothelial-mesenchymal transition in atherosclerosis[J]. Cardiovasc Res, 2018, 114(4):565-577.
21	Lin QQ, Zhao J, Zheng CG, et al. Roles of notch signaling pathway and endothelial-mesenchymal transition in vascular endothelial dysfunction and atherosclerosis[J]. Eur Rev Med Pharmacol Sci, 2018, 22(19):6485-6491.
22	Sun L, Dou FF, Chen JL, et al. Salidroside slows the progression of EA.hy926 cell senescence by regulating the cell cycle in an atherosclerosis model[J]. Mol Med Rep, 2018, 17(1):257-263

[1]	马晓菊, 梁潇, 段云友, 袁丽君, 赵萍. NB_AV脂质纳泡对ApoE ^-/-小鼠动脉粥样硬化病变的评估和干预[J/OL]. 中华医学超声杂志（电子版）, 2024, 21(06): 608-616.
[2]	张凯, 乔永杰, 林志强, 刘健, 邓泽群, 谭飞, 曾健康, 李嘉欢, 李培杰, 周胜虎. 假体周围骨溶解中巨噬细胞极化的机制研究进展[J/OL]. 中华关节外科杂志(电子版), 2024, 18(05): 618-625.
[3]	杨瑾, 刘雪克, 张媛媛, 金钧, 韦瑶. 肠道微生物来源石胆酸对脓毒症相关肝损伤的保护作用[J/OL]. 中华危重症医学杂志(电子版), 2024, 17(04): 265-274.
[4]	杜贵伟, 陆勇, 成博, 贺薏, 梁爽. 钬激光碎石术术后联合坦索罗辛治疗对输尿管结石患者的影响分析[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 491-496.
[5]	高娟, 徐建庆, 闫芳, 丁盛华, 刘霞. Rutkow、TAPP、TEP 手术治疗单侧腹股沟疝患者的临床疗效及对血清炎症因子水平的影响[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(06): 675-680.
[6]	邢嘉翌, 龚佳晟, 祝佳佳, 陆群. 肺癌化疗患者继发肺部感染的病原菌耐药性及炎症因子变化分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 714-718.
[7]	朱军, 宋家伟, 乔一桓, 郭雅婕, 刘帅, 姜玉, 李纪鹏. M2型巨噬细胞特征基因与结肠癌免疫微环境研究[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(04): 303-311.
[8]	李秀玲, 连少锋, 荣刘涛, 李登峰, 饶蕴玉. 利巴韦林联合复方嗜酸乳杆菌治疗轮状病毒肠炎患儿的临床研究[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(04): 369-372.
[9]	石佳娜, 钱琳艳, 姬凯悦, 祁金文, 胡情, 孙佳斌. 从PVAT 白色脂肪棕色化角度探讨中药在防治动脉粥样硬化中的应用[J/OL]. 中华临床医师杂志(电子版), 2024, 18(09): 853-858.
[10]	温绍敏, 王雅晳, 施依璐, 段莎莎, 云书荣, 张小杉. 靶向超声造影技术在动脉粥样硬化治疗中的应用进展[J/OL]. 中华临床医师杂志(电子版), 2024, 18(05): 496-499.
[11]	买买提·依斯热依力, 尹强, 尹海龙, 李治建, 董雨微, 王永康, 克力木·阿不都热依木, 阿吉艾克拜尔·艾萨. 罗乐胃蜜膏抑制酸刺激诱导食管上皮细胞炎症发生的机制研究[J/OL]. 中华胃食管反流病电子杂志, 2024, 11(03): 137-142.
[12]	吴晓明, 翟仰魁, 王娟, 张硕, 许杰, 潘从清. 男性2 型糖尿病患者空腹C 肽和定量胰岛素敏感性检测指数与血浆致动脉粥样硬化指数的相关性[J/OL]. 中华肥胖与代谢病电子杂志, 2024, 10(04): 288-294.
[13]	欧春影, 李晓宾, 郭靖, 朱亮, 许可, 王梦, 安晓雷. 丁苯酞对血管性认知障碍大鼠炎症因子的影响及对认知障碍的改善作用[J/OL]. 中华脑血管病杂志(电子版), 2024, 18(05): 483-487.
[14]	牟磊, 徐东成, 韩鑫, 徐长江, 韩坤锜, 薛叶潇, 牟媛, 秦文玲, 刘相静, 陈哲, 高楠. 五虫通络胶囊防治椎动脉开口支架术后再狭窄发生的效果[J/OL]. 中华脑血管病杂志(电子版), 2024, 18(05): 467-472.
[15]	唐欣, 翟文海, 王润婷, 周胜宇, 靳航. 补体在缺血性卒中疾病中的研究进展[J/OL]. 中华脑血管病杂志(电子版), 2024, 18(04): 382-392.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Mechanism of circulating endothelial microparticles related microRNA in the atherosclerosis induced by inflammatory response of macrophage

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Mechanism of circulating endothelial microparticles related microRNA in the atherosclerosis induced by inflammatory response of macrophage