Galactose up-regulates the level of Siglec-9 glycan ligand in endothelial cells to inhibit macrophage activity

Niting WU; Yuanting SHE; Hongmei LIU; Jin LI; Yi JIA

doi:10.16016/j.2097-0927.202310077

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Galactose up-regulates the level of Siglec-9 glycan ligand in endothelial cells to inhibit macrophage activity

Basic Medicine | 更新时间：2026-01-09

- Galactose up-regulates the level of Siglec-9 glycan ligand in endothelial cells to inhibit macrophage activity
- Journal of Army Medical University Vol. 46, Issue 13, Pages: 1502-1511(2024)
- 作者机构：
  
  400038 重庆，陆军军医大学(第三军医大学)药学与检验医学系药剂学教研室
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81973319)
- DOI：10.16016/j.2097-0927.202310077
  CLC： R343.23;R322.12;R329.25
- Received：20 October 2023，
  
  Revised：2023-11-29，
  
  Published：15 July 2024
- 稿件说明：
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Niting WU, Yuanting SHE, Hongmei LIU, et al. Galactose up-regulates the level of Siglec-9 glycan ligand in endothelial cells to inhibit macrophage activity[J]. Journal of Army Medical University, 2024, 46(13): 1502-1511.
DOI：

Niting WU, Yuanting SHE, Hongmei LIU, et al. Galactose up-regulates the level of Siglec-9 glycan ligand in endothelial cells to inhibit macrophage activity[J]. Journal of Army Medical University, 2024, 46(13): 1502-1511. DOI： 10.16016/j.2097-0927.202310077.

摘要

目的

探究半乳糖对人原代肺动脉内皮细胞上唾液酸特异性Ig样凝集素-9(sialic-acid-binding immunoglobulin-like lectin 9

Siglec-9)聚糖配体表达的影响及作用机制。

方法

采用流式细胞术确定内皮细胞表面Siglec-9聚糖配体的表达并鉴定该聚糖配体末端糖苷键类型；分别用左旋葡萄糖、葡萄糖、N-乙酰基葡萄糖、甘露糖、N-乙酰基甘露糖、半乳糖、唾液酸、蔗糖处理内皮细胞48 h，采用流式细胞术检测内皮细胞Siglec-9聚糖配体的表达；将内皮细胞分为对照组和半乳糖组(

=3)，Western blot、流式细胞术、免疫荧光分析半乳糖对内皮细胞Siglec-9聚糖配体表达的影响；α2-3

8唾液酸酶处理内皮细胞后，Western blot检测半乳糖对内皮细胞Siglec-9聚糖配体恢复时间的影响；半乳糖处理内皮细胞后，Western blot检测分析细胞内唾液酸合成关键酶(GNE、NANS、NANP、CMAS、NPL以及ST3Gals)表达水平，并用RT-qPCR验证相关蛋白的mRNA表达；siRNA敲低NANP、CMAS及ST3Gal-Ⅲ，Western blot检测内皮细胞Siglec-9聚糖配体水平变化；巨噬细胞与内皮细胞共培养实验分析Siglec-9聚糖配体的增加对巨噬细胞的凋亡与吞噬能力的影响。

结果

内皮细胞上表达有Siglec-9聚糖配体且该配体为末端结构为α2-3连接的唾液酸糖蛋白；与对照组比较，半乳糖组内皮细胞上该聚糖配体表达水平显著增加(

＜0.01)，但半乳糖的添加对该配体的自我恢复时间无影响；与对照组比较，半乳糖处理的内皮细胞中NANP、CMAS、ST3Gal-Ⅲ表达水平升高(

＜0.05，

＜0.01)，且RT-qPCR验证结果与其一致；抑制内皮细胞中NANP、CMAS、ST3Gal-Ⅲ的表达后，内皮细胞Siglec-9聚糖配体水平下降(

＜0.01)。共培养实验中，与未处理组比较，半乳糖处理的内皮细胞促进巨噬细胞凋亡增加(

＜0.01)，并导致其吞噬能力减弱(

＜0.05)。

结论

半乳糖通过NANP-CMAS-ST3Gal-Ⅲ通路上调内皮细胞上Siglec-9聚糖配体水平，从而抑制巨噬细胞活性。

Abstract

Objective

To investigate the effect and mechanism of galactose on the expression of sialic-acid-binding immunoglobulin-like lectin 9 (Siglec-9) glycan ligand on human primarily cultured pulmonary artery endothelial cells.

Methods

The expression of Siglec-9 glycan ligand on the endothelial cell surface and the type of glycosidic bonds at the end of this ligand were identified by flow cytometry. Endothelial cells were treated with L-glucose

glucose

N-acetylglucose

mannose

N-acetylmannose

galactose

sialic acid and sucrose for 48 h

and the expression of Siglec-9 glycan ligand in endothelial cells was detected by flow cytometry. The endothelial cells were divided into the control group and the galactose group (

=3). Western blotting

flow cytometry

and immunofluorescence assay were employed to investigate the impact of galactose on the Siglec-9 glycan ligand in endothelial cells. After α2-3

8 sialidase treatment for endothelial cells

Western blotting was used to detected the effect of galactose on the recovery time of Siglec-9 glycan ligand in endothelial cells. Endothelial cells were treated with galactose

Western blotting was used to detect and analyze the expression levels of intracellular sialic acid synthetases (GNE

NANS

NANP

CMAS

NPL and ST3Gals)

and the mRNA expression levels of the relevant proteins were verified by RT-qPCR. Western blotting was used to detect the changes of Siglec-9 glycan ligand level in endothelial cells after siRNA knockdown in NANP

CMAS and ST3Gal-Ⅲ. The effect of the increase of Siglec-9 ligand on apoptosis and phagocytosis of macrophages was analyzed by macrophage co-culture experiments.

Results

Endothelial cells showed the expression of Siglec-9 ligand and the ligand was a sialic acid glycoprotein linked to α 2-3 sialic acid at the end. Compared with the control group

the express

ion level of this ligand on endothelial cells in the galactose group was increased significantly (

0.01)

but the addition of galactose had no effect on the self-recovery time of the ligand. Compared with the control group

the expression levels of NANP

CMAS and ST3Gal-Ⅲ in endothelial cells treated with galactose were increased (

0.05

0.01)

respectively. The results of RT-qPCR verification were consistent with those results. After the expression of NANP

CMAS or ST3Gal-Ⅲ was inhibited

the level of Siglec-9 glycan ligand in endothelial cells was decreased (

0.01). In the co-culture experiment

compared with the untreated group

galactose-treated endothelial cells promoted the apoptosis of macrophages (

0.01) and reduced their phagocytosis (

0.05).

Conclusion

Galactose up-regulates the level of Siglec-9 glycan ligand on endothelial cells through the NANP-CMAS-ST3Gal-Ⅲ pathway

thereby inhibiting the activity of macrophages.

关键词

Keywords

references

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