Jagged1/Notch1信号通路对特发性肺纤维化过程中内皮间质转化的影响研究

doi:10.12114/j.issn.1007-9572.2023.0766

中国全科医学 ›› 2025, Vol. 28 ›› Issue (25): 3151-3160.DOI: 10.12114/j.issn.1007-9572.2023.0766

所属专题：呼吸疾病文章合辑

Jagged1/Notch1信号通路对特发性肺纤维化过程中内皮间质转化的影响研究

杨其芬¹, 赵惠亮², 郭永胜¹, 渠景连¹^,^*()

1．550025　贵州省贵阳市，贵州中医药大学基础医学院
2．550025　贵州省贵阳市，贵州中医药大学人文与管理学院

收稿日期:2024-07-15 修回日期:2024-12-30 出版日期:2025-09-05 发布日期:2025-07-24
通讯作者: 渠景连
作者贡献：
杨其芬提出主要研究目标，负责研究的构思、设计与实施，进行数据的收集与整理、统计学处理，图表的绘制与展示，撰写论文；赵惠亮、郭永胜进行论文的修订；渠景连负责文章的质量控制与审查，监督管理，对文章整体负责。
基金资助:
国家自然科学基金资助项目(81660789，82160892)

The Impact of the Jagged1/Notch1 Signalling Pathway on Endothelial-mesenchymal Transition in Idiopathic Pulmonary Fibrosis

YANG Qifen¹, ZHAO Huiliang², GUO Yongsheng¹, QU Jinglian¹^,^*()

1. School of Basic Medicine, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China
2. College of Humanities and Management, Guizhou University of Traditional Chinese Medicine, Guiyang 550025, China

Received:2024-07-15 Revised:2024-12-30 Published:2025-09-05 Online:2025-07-24
Contact: QU Jinglian

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摘要/Abstract

摘要： 背景特发性肺纤维化（IPF）是一种病理机制复杂且尚无有效治疗手段的世界性疾病，内皮间质转化（EndMT）作为多种慢性血管疾病的病理基础，近年来发现也是IPF的重要危险因素之一，但其相关机制却尚未明确。目的观察Jagged1/Notch1信号通路对EndMT的影响，以阐明IPF过程中EndMT的相关机制。方法 2021年3—6月体外培养人肺动脉内皮细胞（HPAEC），取HPAEC加空白血清作为对照组，5 mg/L TGF-β1刺激HPAEC 72 h作为模型组，造模后加500 nmol/L Notch信号阻断剂（DAPT）作为DAPT组。采用反转录·聚合酶链反应（RT-PCR）检测Notch1、Jagged1和启动子结合因子（CBF1）mRNA的表达，蛋白质印迹法（WB）检测Notch1，Jagged1和CBF1蛋白的表达，免疫共沉淀法（Co-IP）检测Notch1活性胞内结构域蛋白（NICD1）与CBF1的结合情况，噻唑蓝比色法（MTT）检测细胞增殖情况，Transwell和划痕试验检测细胞迁移能力，免疫荧光检测内皮细胞标志物血小板内皮细胞黏附分子1（CD31）和血管内皮钙粘蛋白（VE-cadherin）以及间质细胞标志物成纤维细胞特异性蛋白1（FSP1）和α-平滑肌肌动蛋白（α-SMA）的表达。结果 3组Notch1、Jagged1和CBF1 mRNA和蛋白表达水平、细胞增殖和迁移能力比较，差异均有统计学意义（P<0.05）。其中空白组和DAPT组Notch1、Jagged1和CBF1 mRNA和蛋白表达、细胞增殖和迁移能力均低于模型组（P<0.05）。Co-IP实验结果显示，3组NICD1与CBF1的结合情况有差异，其中空白组和DAPT组NICD1与CBF1的结合受到抑制，模型组NICD1与CBF1发生结合。免疫荧光结果显示，3组CD31、VE-Cadherin、FSP1和α-SMA表达比较，差异均有统计学意义（P<0.05）；其中空白组和DAPT组内皮细胞标志物CD31、VE-Cadherin表达高于模型组，间质细胞标志物FSP1、α-SMA表达低于模型组（P<0.05）。结论 Jagged1/Notch1信号通路参与了IPF过程中的EndMT进程，阻断该信号通路可抑制EndMT。

关键词: 特发性肺纤维化, 内皮细胞, 内皮间质转化, Jagged1/Notch1信号通路, 人肺动脉内皮细胞

Abstract:

Background

Idiopathic pulmonary fibrosis (IPF) is a complex global disease with a pathological mechanism that remains poorly understood and for which there is currently no effective treatment. Endothelial-mesenchymal transition (EndMT) , identified as a pathological basis for various chronic vascular diseases, has recently been recognised as a significant risk factor for IPF. However, the related mechanisms have yet to be clarified.

Objective

To observe the effects of the Jagged1/Notch1 signalling pathway on EndMT to elucidate the related mechanisms during IPF.

Methods

From March to June 2021, human pulmonary arterial endothelial cells (HPAEC) were cultured in vitro. A control group was established using HPAEC with blank serum, while a model group was created by stimulating HPAEC with 5 mg/L TGF-β₁ for 72 hours. In the DAPT group, the same TGF-β₁ stimulation was followed by the addition of 500 nmol/L Notch signalling inhibitor for 72 hours. Reverse transcription quantitative polymerase chain reaction (RT-PCR) was employed to measure the expression of Notch1, Jagged1, and the CBF1 Promoter (CBF1) mRNA, while Western blotting (WB) was used to assess the protein expressions of Notch1, Jagged1, and CBF1. Co-immunoprecipitation (Co-IP) was conducted to examine the binding of Notch1 intracellular domain protein (NICD1) with CBF1, and the Methylthiazoltetrazolium Colorimetry (MTT) assay was performed to evaluate cell proliferation. Transwell and scratch assays were conducted to measure cell migration ability, and immunofluorescence was used to detect the expression of endothelial cell markers CD₃₁ and VE-cadherin, as well as mesenchymal cell markers fibroblast-specific protein 1 (FSP1) and α-smooth muscle actin (α-SMA) .

Results

Statistical analysis revealed significant differences (P<0.05) in the expression levels of Notch1, Jagged1, CBF1 mRNA and proteins, as well as cell proliferation and migration abilities among the three groups. The blank group and DAPT group exhibited lower expressions of Notch1, Jagged1, and CBF1 mRNA and proteins, alongside reduced cell proliferation and migration compared to the model group, with significant differences noted (P<0.05) . Co-IP results indicated discrepancies in the binding of NICD1 and CBF1 among the three groups, with binding inhibited in the blank and DAPT groups, while binding occurred in the model group. Immunofluorescence results demonstrated differences in the expressions of CD₃₁, VE-Cadherin, FSP1, and α-SMA among the 3 groups, where the blank and DAPT groups showed higher expressions of endothelial markers CD₃₁ and VE-Cadherin compared to the model group, while FSP1 and α-SMA expressions were lower.

Conclusion

The Jagged1/Notch1 signalling pathway is involved in the EndMT process during IPF, and inhibiting this signalling pathway can suppress EndMT.

Key words: Idiopathic pulmonary fibrosis, Endothelial cells, Endothelial-to-mesenchymal transition, Jagged1/Notch1 signaling pathway, Human pulmonary artery endothelial cells

中图分类号:

R 563.9

杨其芬,赵惠亮,郭永胜,等. Jagged1/Notch1信号通路对特发性肺纤维化过程中内皮间质转化的影响研究[J]. 中国全科医学, 2025, 28(25): 3151-3160. DOI: 10.12114/j.issn.1007-9572.2023.0766.
YANG Qifen,ZHAO Huiliang,GUO Yongsheng, et al. The Impact of the Jagged1/Notch1 Signalling Pathway on Endothelial-mesenchymal Transition in Idiopathic Pulmonary Fibrosis[J]. Chinese General Practice, 2025, 28(25): 3151-3160. DOI: 10.12114/j.issn.1007-9572.2023.0766.

图/表 14

表1 RT-PCR引物序列

Table 1 Primer sequence of RT-PCR

基因	引物方向（5'→3'）
GAPDH	5'-CAGGGCTGCTTTTAACTCTGGTAA-3' 5'-GGGTGGAATCATATTGGAACATGT-3'
Notch1	5'-GGGACCAACTGTGACATCAA-3' 5'-GTAGCCACTGGTCATGTCTTT-3'
Jagged1	5'-TCAACGGGGGAACTTGTAGC-3' 5'-CCTGGGGAACACTCACACTC-3'
CBF1	5'-TCATGCCAGTTCACAGCAGT-3' 5'-GGAGTGCCATGCCAGTAACT-3'

表2 各组Notch 1、Jagged 1和CBF1 mRNA表达水平（±s，n=3）

Table 2 The expression levels of Notch 1，Jagged 1 and CBF1 mRNA in each group

组别	Notch1 mRNA	Jagged1 mRNA	CBF1 mRNA
空白组	1.000±0.058	1.000±0.014	1.000±0.038
模型组	3.182±0.050^a	2.329±0.078^a	2.187±0.039^a
DAPT组	1.540±0.218^ab	1.437±0.032^b	1.486±0.070^ab
F值	119.584	468.715	368.121
P值	<0.001	<0.001	<0.001

表3 各组Notch 1、Jagged 1和CBF1蛋白条带灰度值（±s，n=3）

Table 3 The gray values of Notch 1，Jagged 1 and CBF1 protein band in each group

组别	Notch1/GAPDH	Jagged1/GAPDH	CBF1/GAPDH
空白组	0.677±0.033	0.292±0.023	0.250±0.026
模型组	1.444±0.038^a	0.717±0.032^a	0.737±0.022^a
DAPT组	0.966±0.004^ab	0.453±0.018^ab	0.509±0.013^ab
F值	519.591	209.932	378.314
P值	<0.001	<0.001	<0.001

图1 各组Notch 1、Jagged 1和CBF1蛋白条带图注：GAPDH=甘油醛-3-磷酸脱氢酶，Notch1=Notch受体1，CBF1=启动子结合因子，DAPT=Jagged1/Notch1信号阻断剂。

Figure 1 Bands of Notch 1，Jagged 1 and CBF1 in each group

图2 各组NICD1与CBF1的结合情况注：NICD1=Notch1活性胞内结构域蛋白。

Figure 2 The combination of NICD1 and CBF1 in each group

表4 各组的吸光度（±s，n=3）

Table 4 Absorbances in each group

组别	24 h		48 h		72 h
组别	细胞增殖（A）	抑制率（%）	细胞增殖（A）	抑制率（%）	细胞增殖（A）	抑制率（%）
空白组	0.446±0.024	—	0.724±0.031	—	0.783±0.037	—
模型组	0.472±0.019	—	0.926±0.016^a	—	1.325±0.018^a	—
DAPT组	0.441±0.014^b	6.567	0.793±0.013^ab	14.362	0.844±0.046^ab	36.301
F值	3.594	—	109.493	—	338.369	—
P值	0.060	—	<0.001	—	<0.001	—

表5 各组的迁移细胞数和迁移率（±s，n=3）

Table 5 The number and mobility of migrating cells in each group

组别	细胞数	迁移率（%）
空白组	41.00±7.55	100.00
模型组	76.67±8.62^a	186.99
DAPT组	52.33±4.04^b	127.64
F值	20.242	—
P值	0.002	—

图3 各组Transwell实验的结果（Transwell，×400）注：A代表空白组，B代表模型组，C代表DAPT组。

Figure 3 Results of Transwell in each group（Transwell，×400）

表6 各组的划痕距离（±s，n=3，μm）

Table 6 Scratch distances in each group

组别	0 h	12 h	24 h
空白组	220.000±3.606	169.000±2.000	91.667±7.234
模型组	214.667±1.528	96.333±5.507^a	51.333±3.055^a
DAPT组	213.000±4.359	130.667±10.692^ab	70.000±8.185^ab
F值	3.305	79.998	28.500
P值	0.098	<0.001	0.001

图4 各组划痕试验的结果（划痕试验，×100）

Figure 4 The results of the scratch assays in each group（SA，×100）

图5 各组CD31的表达（免疫荧光，×200）

Figure 5 Expression of CD31 in each group（IF，×200）

图6 各组VE-Cadherin的表达（免疫荧光，×200）

Figure 6 Expression of VE-Cadherin in each group（IF，×200）

图7 各组FSP1的表达（免疫荧光，×200）

Figure 7 Expression of FSP1 in each group（IF，×200）

图8 各组α-SMA的表达（免疫荧光，×200）注：α-SMA=α-平滑肌肌动蛋白。

Figure 8 Expression of α-SMA in each group（IF，×200）

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Jagged1/Notch1信号通路对特发性肺纤维化过程中内皮间质转化的影响研究

The Impact of the Jagged1/Notch1 Signalling Pathway on Endothelial-mesenchymal Transition in Idiopathic Pulmonary Fibrosis

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