oxLDL/β2GPⅠ/aβ2GPⅠ复合物通过TLR4/MyD88/NF-κB途径促进血管内皮细胞血管生成

doi:10.11958/20240680

天津医药 ›› 2024, Vol. 52 ›› Issue (11): 1131-1136.doi: 10.11958/20240680

oxLDL/β2GPⅠ/aβ2GPⅠ复合物通过TLR4/MyD88/NF-κB途径促进血管内皮细胞血管生成

张贵婷¹(), 何超²^,^△()

1 南京大学医学院附属鼓楼医院检验科（邮编210008）
2 江苏大学第四附属医院中心实验室

收稿日期:2024-05-29 修回日期:2024-07-10 出版日期:2024-11-15 发布日期:2024-11-12
通讯作者: △E-mail：15952853808@163.com
作者简介:张贵婷（1991），女，技师，主要从事抗磷脂综合征实验室诊断及发病机制研究。E-mail：chujianzgt@163.com
基金资助:
江苏省卫生健康委科研项目(H2023033);镇江市重点研发计划社会发展项目(SH2022082)

Mechanism of oxLDL/β2GPⅠ/aβ2GPⅠ complex promoting the angiogenesis in vascular endothelial cells through TLR4//MyD88/NF-κB signaling pathway

ZHANG Guiting¹(), HE Chao²^,^△()

1 Department of Laboratory Medicine, Nanjing Drum Tower Hospital Clinical College of Nanjing University, Nanjing 210008, China
2 Center Laboratory, the Fourth Affiliated Hospital of Jiangsu University

Received:2024-05-29 Revised:2024-07-10 Published:2024-11-15 Online:2024-11-12
Contact: △E-mail：15952853808@163.com

张贵婷, 何超. oxLDL/β2GPⅠ/aβ2GPⅠ复合物通过TLR4/MyD88/NF-κB途径促进血管内皮细胞血管生成[J]. 天津医药, 2024, 52(11): 1131-1136.

ZHANG Guiting, HE Chao. Mechanism of oxLDL/β2GPⅠ/aβ2GPⅠ complex promoting the angiogenesis in vascular endothelial cells through TLR4//MyD88/NF-κB signaling pathway[J]. Tianjin Medical Journal, 2024, 52(11): 1131-1136.

摘要/Abstract

摘要：

目的探讨氧化型低密度脂蛋白（oxLDL）/β2糖蛋白Ⅰ（β2GPⅠ）/抗β2糖蛋白Ⅰ抗体（aβ2GPⅠ）复合物对血管内皮细胞增殖、迁移及血管生成的影响及其机制。方法将人脐静脉内皮细胞（HUVEC）培养至对数生长期，分为对照组（正常培养）、oxLDL组（50 mg/L oxLDL）、oxLDL/β2GPⅠ/aβ2GPⅠ组（50 mg/L oxLDL/100 mg/L β2GPⅠ/100 mg/L aβ2GPⅠ）和血管内皮生长因子（VEGF）组（100 μg/L VEGF）。实时荧光定量PCR（qPCR）法检测血管生成相关因子VEGF、血管内皮钙黏蛋白（VE-cadherin）、基质金属蛋白酶（MMP）-2及MMP-9的基因表达；CCK-8检测细胞增殖；划痕愈合实验和Transwell实验检测细胞的迁移和侵袭能力；基质胶管腔形成实验检测HUVEC的血管生成能力；Western blot法检测Toll样受体4（TLR4）、髓样分化因子88（MyD88）和核因子κB（NF-κB）的蛋白表达。结果与对照组相比，oxLDL/β2GPⅠ/aβ2GPⅠ组细胞增殖活力在处理48 h时增加；此外，与对照组相比，oxLDL/β2GPⅠ/aβ2GPⅠ组细胞迁移及血管生成能力增强，VEGF、VE-cadherin、MMP-2及MMP-9的mRNA水平升高（P<0.05），TLR4和MyD88蛋白水平及p-NF-κB p65/NF-κB p65比值升高（P<0.05）。结论 oxLDL/β2GPⅠ/aβ2GPⅠ复合物可能通过TLR4/MyD88/NF-κB通路诱导血管内皮细胞增殖、迁移及血管生成。

关键词: 动脉粥样硬化, β2糖蛋白Ⅰ, 新生血管化, 病理性, 细胞增殖, 细胞运动, NF-κB, 内皮细胞, 氧化型低密度脂蛋白

Abstract:

Objective To investigate effects of oxidized low density lipoprotein/β2 glycoprotein-Ⅰ/anti-β2 glycoprotein-Ⅰ antibody (oxLDL/β2GPⅠ/aβ2GPⅠ) complex on the proliferation, migration and angiogenesis of vascular endothelial cells and its mechanism. Methods Human umbilical vein endothelial cells (HUVEC) were cultured to logarithmic growth phase and grouped into the control group (normal culture), the oxLDL group (50 mg/L oxLDL), the oxLDL/β2GPⅠ/aβ2GPⅠ group (50 mg/L oxLDL/100 mg/L β2GPⅠ/100 mg/L aβ2GPⅠ) and the VEGF group (100 μg/L VEGF). The gene expressions of VEGF, vascular endothelial cadherin (VE-cadherin), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by real-time quantitative fluorescent PCR (qPCR). Cell counting kit-8 (CCK-8) method was employed to detect cell proliferation. Cell migration and invasion were determined by scratch healing test and Transwell assay. Matrigel tube formation assay was used to observe the angiogenesis of HUVEC. The relative protein expression of TLR4, MyD88 and NF-κB were examined by Western blot assay. Results Compared with the control group, the proliferation activity of cells at 48 h of treatment was increased in the oxLDL/β2GPⅠ/aβ2GPⅠ group (P<0.05). Moreover, compared with the control group, cell migration and angiogenesis were increased in the oxLDL/β2GPⅠ/aβ2GPⅠ group, and the mRNA levels of VEGF, VE-cadherin, MMP-2 and MMP-9 were elevated (P<0.05). Compared with the control group, levels of TLR4 and MyD88 were elevated in the oxLDL/β2GPⅠ/aβ2GPⅠ complex group (P<0.05), as well as levels of p-NF-κB p65/NF-κB p65 (P<0.05). Conclusion oxLDL/β2GPⅠ/aβ2GPⅠ complex may promote the proliferation, migration and tube formation of vascular endothelial cells by regulating TLR4/MyD88/NF-κB signaling pathway.

Key words: atherosclerosis, beta 2-glycoproteinⅠ, neovascularization, pathologic, cell proliferation, cell movement, NF-kappa B, endothelial cells, oxidized low density lipoprotein

中图分类号:

R392.9

图/表 10

表1 qPCR引物序列

Tab.1 Sequences of qPCR primers

基因名称	引物序列（5'→3'）	产物大小/bp
VEGF	上游：AGCTTCGTGTCCTGTATGGC	138
VEGF	下游：TTTCTGGCCACGTCCAGTTT	138
VE-cadherin	上游：CCCACTATCCGATACGAATACC	149
VE-cadherin	下游：ATCCACATCTAGGACGTTGATG	149
MMP-2	上游：TCAAGTTCCCCGGCGATG	225
MMP-2	下游：AGTTGGCCACATCTGGGTTG	225
MMP-9	上游：CAAAGACCTGAAAACCTCCAAC	168
MMP-9	下游：GACTGCTTCTCTCCCATCATC	168
β-actin	上游：GCTTGCGGCATCCACGAGAC	82
β-actin	下游：CGGTGTTGGCATACAGATCCTTACG	82

表2 3组HUVEC细胞不同时间点增殖情况比较（n=5，OD450值，$\bar{x} \pm s$s）

Tab.2 Comparison of HUVEC cell proliferation at different time points between three groups

组别	0 h	24 h	48 h	72 h	96 h
对照组	1.00±0.05	1.43±0.14	1.81±0.32	4.15±0.22	5.28±0.34
oxLDL组	0.85±0.42	1.60±0.14	2.70±0.26	4.17±0.16	5.47±0.18
oxLDL/β2GPⅠ/aβ2GPⅠ组	1.05±0.03	1.29±0.18^b	2.40±0.22^a	3.78±0.22^ab	5.54±0.42
F	0.870	4.945^＊	14.040^＊＊	6.025^＊	0.858

图1 各组HUVEC细胞迁移情况（×100）

Fig.1 Migration of HUVEC cells in four groups (×100)

图2 各组HUVEC细胞侵袭情况（结晶紫染色，×200）

Fig.2 Invasion of HUVEC cells in four groups (crystal violet staining, ×200)

表3 各组HUVEC细胞迁移、侵袭及血管生成的比较（n=3，$\bar{x} \pm s$）

Tab.3 Comparison of migration, invasion and tube formation between four groups of HUVEC cells

组别	迁移率/%	侵袭细胞数量/个	管腔节点/%
对照组	100.00±20.78	65.33±18.90	100.00±16.33
oxLDL组	168.70±85.09	173.70±16.50	231.50±55.22
oxLDL/β2GPⅠ/ aβ2GPⅠ组	421.80±83.91^ab	224.70±11.68^ab	369.60±65.47^a
VEGF组	355.00±70.66^ab	192.00±14.73^a	392.90±59.86^ab
F	14.030^＊＊	58.160^＊＊	19.800^＊＊

图3 各组HUVEC管腔形成情况（钙黄绿素染色，×200）

Fig.3 Tube formation of HUVEC cells in four groups (Calcein AM staining, ×200)

表4 3组HUVEC细胞VEGF、VE-cadherin、MMP-2及MMP-9 mRNA表达水平比较（n=4，$\bar{x} \pm s$）

Tab.4 Comparison of mRNA expression levels of VEGF, VE-cadherin, MMP-2 and MMP-9 in HUVEC between three groups

组别	VEGF	VE-cadherin	MMP-2	MMP-9
对照组	1.00±0.05	1.00±0.30	1.00±0.03	1.00±0.12
oxLDL组	1.20±0.22	3.77±0.36	1.32±0.35	2.07±0.16
oxLDL/β2GPⅠ/ aβ2GPⅠ组	1.79±0.46^a	4.71±0.31^ab	2.41±0.46^ab	2.21±0.43^a
F	7.587^＊	140.900^＊＊	19.030^＊＊	23.150^＊＊

图4 2组HUVEC细胞中TLR4和MyD88蛋白表达 A：对照组；B：oxLDL/β2GPⅠ/aβ2GPⅠ组。

Fig.4 The protein expression levels of TLR4 and MyD88 in HUVEC cells of two groups

表5 2组HUVEC细胞中TLR4、MyD88和p-NF-κB 蛋白表达水平比较（n=3，$\bar{x} \pm s$）

Tab.5 Comparison of protein expression levels of TLR4, MyD88, p-NF-κB p65 in HUVEC between two groups

组别	TLR4	MyD88	p-NF-κB p65/NF-κB p65
对照组	1.00±0.14	1.00±0.18	1.00±0.13
oxLDL/β2GPⅠ/ aβ2GPⅠ组	1.62±0.32	2.29±0.45	1.86±0.23
t	3.088^＊	4.570^＊	5.708^＊＊

图5 2组HUVEC细胞中p-NF-κB p65/NF-κB p65蛋白表达 A：对照组；B：oxLDL/β2GPⅠ/aβ2GPⅠ组。

Fig.5 The protein expression levels of p-NF-κB p65/NF-κB p65 in HUVEC cells in two groups

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oxLDL/β2GPⅠ/aβ2GPⅠ复合物通过TLR4/MyD88/NF-κB途径促进血管内皮细胞血管生成

Mechanism of oxLDL/β2GPⅠ/aβ2GPⅠ complex promoting the angiogenesis in vascular endothelial cells through TLR4//MyD88/NF-κB signaling pathway

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