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

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

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