基于NOD样受体3炎性小体通路对利拉鲁肽在氧化低密度脂蛋白诱导内皮细胞损伤的作用机制研究

doi:10.12114/j.issn.1007-9572.2023.0531

摘要/Abstract

摘要： 背景动脉粥样硬化是世界范围内引起心脑血管疾病最主要的原因，炎症是目前研究热点，其中NOD样受体3（NLRP3）是研究最为深入的炎症小体。胰高糖素样肽1（GLP-1）受体激动剂有抗动脉粥样硬化作用，具体机制尚不明确。目的研究利拉鲁肽通过拮抗氧化低密度脂蛋白（ox-LDL）诱导的内皮细胞损伤的作用机制。方法 2022-03-25—05-19培养人脐静脉内皮细胞（HUVEC），取HUVEC加空白血清作为对照组，100 μg/mL的ox-LDL干预HUVEC 48 h作为模型组，100 μg/mL的ox-LDL干预HUVEC 24 h后分别加入100、200、400 nmol/L利拉鲁肽处理24 h作为利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组。CCK-8法计算细胞增殖率。通过扫描电镜观察焦亡细胞形态。检测乳酸脱氢酶（LDH）活力。酶联免疫吸附试验（ELISA）检测白介素（IL）-1β、IL-18表达水平。蛋白质免疫印迹试验（Western blot）检测NLRP3、接头蛋白凋亡相关斑点样蛋白（ASC）、天冬氨酸蛋白水解酶1（Caspase-1）、焦亡执行蛋白（GSDMD）、N端结构域的焦亡执行蛋白（N-GSDMD）表达水平。结果模型组、利拉鲁肽低浓度组和利拉鲁肽中浓度组细胞增殖率低于对照组，利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组细胞增殖率高于模型组（P<0.05）。细胞扫描电镜结果示模型组细胞焦亡明显，利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组细胞焦亡情况明显改善。模型组、利拉鲁肽低浓度组LDH活力高于对照组，利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组低于模型组（P<0.05）。模型组、利拉鲁肽低浓度组IL-1β表达水平高于对照组，利拉鲁肽中浓度组、利拉鲁肽高浓度组IL-1β表达水平低于模型组（P<0.05）；模型组IL-18表达水平高于对照组，利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组IL-18表达水平低于模型组（P<0.05）。模型组NLRP3、ASC、Caspase-1、GSDMD、N-GSDMD表达水平高于对照组，利拉鲁肽低浓度组ASC、Caspase-1表达水平高于对照组，利拉鲁肽中浓度组NLRP3、ASC表达水平低于模型组，利拉鲁肽高浓度组NLRP3、ASC、Caspase-1表达水平低于模型组（P<0.05）。结论利拉鲁肽显著抑制ox-LDL诱导的内皮细胞NLRP3炎性小体活化，并且能够抑制内皮细胞的焦亡，具有抗动脉粥样硬化作用。

关键词: 动脉粥样硬化, 利拉鲁肽, 内皮细胞, 氧化低密度脂蛋白, NOD样受体3

Abstract:

Background

Atherosclerosis is the primary cause of cardiovascular and cerebrovascular diseases worldwide, and inflammation is a current research focus, with NOD-like receptor 3 (NLRP3) being the most intensively studied inflammasome. GLP-1 receptor agonists have shown anti-atherosclerotic effects, but the underlying mechanisms remain unclear.

Objective

To investigates the mechanism of liraglutide in antagonizing oxidized low-density lipoprotein (ox-LDL) induced endothelial cell injury.

Methods

From March 25 to May 19, 2022, Human umbilical vein endothelial cells (HUVEC) were cultured, and HUVEC with blank serum was served as the control group, 100 μg/mL ox-LDL treated HUVEC for 48 hours was served as the model group. Liraglutide was added in concentrations of 100 nmol/L, 200 nmol/L, and 400 nmol/L to the HUVECs treated with ox-LDL for 24 hours, forming low, medium, and high concentration liraglutide groups, respectively. Cell proliferation rates were calculated using the CCK-8 method. Pyroptotic cell morphology was observed by using scanning electron microscopy. Lactate dehydrogenase (LDH) activity was measured. The expression levels of interleukin (IL) -1β and IL-18 were detected using enzyme-linked immunosorbent assay (ELISA) . Western blot was used to assess the expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) , caspase-1, gasdermin D (GSDMD) , and N-terminal GSDMD (N-GSDMD) .

Results

Cell proliferation rate in the model group and both low and medium concentration liraglutide groups were lower than the control group, while the rate in all liraglutide-treated groups were higher than the model group (P<0.05) . Scanning electron microscopy showed obvious pyroptosis in the model group cells, which was significantly reduced in all liraglutide-treated groups. LDH activity in the model group and the low concentration liraglutide group was higher than the control group, while it was lower in all liraglutide-treated groups compared to the model group (P<0.05) . IL-1β level in the model group and the low concentration liraglutide group was higher than the control group, whereas IL-1β levels in the medium and high concentration liraglutide groups was lower than the model group (P<0.05) . IL-18 level in the model group was higher than the control group, while level in all liraglutide-treated groups was lower than the model group (P<0.05) . The expression levels of NLRP3, ASC, Caspase-1, GSDMD, and N-GSDMD in the model group were higher than the control group. In the low concentration liraglutide group, ASC and Caspase-1 levels were higher than the control group, whereas in the medium concentration group, NLRP3 and ASC levels were lower than the model group. In the high concentration group, NLRP3, ASC, and Caspase-1 levels were lower than the model group (P<0.05) .

Conclusion

Liraglutide significantly inhibits NLRP3 inflammasome activation in endothelial cells induced by ox-LDL, and can inhibit endothelial cell pyroptosis, with anti-atherosclerotic effects.

Key words: Atherosclerosis, Liraglutide, Endothelial cells, Oxidized low-density lipoprotein, NOD-like receptor 3

中图分类号:

R 543.5

陈玲,徐锐,程新春,等. 基于NOD样受体3炎性小体通路对利拉鲁肽在氧化低密度脂蛋白诱导内皮细胞损伤的作用机制研究[J]. 中国全科医学, 2025, 28(05): 601-606. DOI: 10.12114/j.issn.1007-9572.2023.0531.
CHEN Ling,XU Rui,CHENG Xinchun, et al. Mechanism of Liraglutide in Oxidized Low-density Lipoprotein Induced Endothelial Cell Injury Based on NOD-like Receptor 3 Inflammasome Pathway[J]. Chinese General Practice, 2025, 28(05): 601-606. DOI: 10.12114/j.issn.1007-9572.2023.0531.

图/表 6

表1 5组HUVEC细胞增殖率比较（±s，%，n=5）

Table 1 Comparison of the proliferation rates of 5 groups of HUVEC cells

分组	细胞增殖率
对照组	100.00
模型组	40.75±4.11^a
利拉鲁肽低浓度组	53.16±4.78^ab
利拉鲁肽中浓度组	60.41±4.97^ab
利拉鲁肽高浓度组	90.20±9.77^b
F值	99.02
P值	<0.001

图1 各组细胞电镜扫描结果注：图A~E分别为对照组、模型组、利拉鲁肽低浓度组、利拉鲁肽中浓度组、利拉鲁肽高浓度组。

Figure 1 Electron microscope scanning results of each cell group

表2 各组细胞LDH活力（±s，U/mL，n=3）

Table 2 LDH activity of cells in each group

分组	LDH活力
对照组	60.67±1.94
模型组	81.04±6.71^a
利拉鲁肽低浓度组	71.21±2.31^ab
利拉鲁肽中浓度组	68.79±2.51^b
利拉鲁肽高浓度组	65.39±1.82^b
F值	13.63
P值	<0.001

表3 各组细胞IL-1β、IL-18表达水平（±s，pg/mL，n=3）

Table 3 The expression levels of IL-1β and IL-18 in each group

分组	IL-1β	IL-18
对照组	56.57±2.87	170.05±9.60
模型组	71.34±3.09^a	225.64±14.22^a
利拉鲁肽低浓度组	64.38±2.84^a	195.94±13.45^b
利拉鲁肽中浓度组	58.91±2.62^b	184.19±18.45^b
利拉鲁肽高浓度组	56.24±2.86^b	163.14±10.17^b
F值	15.02	9.93
P值	<0.001	0.002

表4 各组细胞NLRP3炎性小体活化相关蛋白与焦亡相关蛋白条带灰度值（±s，n=3）

Table 4 The gray values of NLRP3 inflammasome activation related protein and pyrodeath related protein band in each group

分组	NLRP3	ASC	Caspase-1	GSDMD	N-GSDMD
对照组	0.59±0.08	0.64±0.04	0.73±0.19	0.82±0.07	0.81±0.15
模型组	0.93±0.06^a	1.22±0.23^a	1.40±0.22^a	1.35±0.20^a	1.24±0.21^a
利拉鲁肽低浓度组	0.80±0.06	1.03±0.09^a	1.27±0.13^a	1.14±0.13	1.12±0.12
利拉鲁肽中浓度组	0.74±0.11^b	0.71±0.03^b	1.04±0.16	1.06±0.13	1.02±0.13
利拉鲁肽高浓度组	0.70±0.10^b	0.65±0.04^b	0.91±0.08^b	1.06±0.19	0.94±0.13
F值	6.934	16.190	8.207	4.924	3.589
P值	0.006	0.001	0.003	0.019	0.046

图2 各组细胞NLRP3炎性小体活化相关蛋白与焦亡相关蛋白条带图注：NLRP3=NOD样受体3，ASC=接头蛋白凋亡相关斑点样蛋白，Caspase-1=天冬氨酸蛋白水解酶1，GSDMD=焦亡执行蛋白，N-GSDMD=抗N端结构域的焦亡执行蛋白，β-actin=肌动蛋白。

Figure 2 Bands of NLRP3 inflammasome activation related proteins and pyroptosis related proteins in each group of cells

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