S1P/S1PR2信号轴通过阻断自噬流促进心肌成纤维细胞活化与胶原分泌而恢复自噬流可逆转纤维化

刘佳; 玛依热·阿吉; 谢耀慧; 王旭敏

doi:10.16016/j.2097-0927.202608001

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S1P/S1PR2信号轴通过阻断自噬流促进心肌成纤维细胞活化与胶原分泌而恢复自噬流可逆转纤维化

基础医学 | 更新时间：2026-10-30

- S1P/S1PR2信号轴通过阻断自噬流促进心肌成纤维细胞活化与胶原分泌而恢复自噬流可逆转纤维化
- S1P/S1PR2 axis promotes cardiac fibroblast activation and collagen secretion by blocking autophagic flux, while restoring autophagic flux reverses fibrosis
- 陆军军医大学学报 2026年48卷第20期页码：2908-2919
- 作者机构：
  
  新疆医科大学第六附属医院心血管内科，新疆乌鲁木齐
- 作者简介：
  
  王旭敏，E-mail：wangxuminsparky@163.com
- 基金信息：
  
  新疆维吾尔自治区自然科学基金面上项目(2021D01C457;2024D01C146)
- DOI：10.16016/j.2097-0927.202608001
  中图分类号： R322.11;R329.26;R363.21
- 收稿：2026-08-02，
  
  修回：2026-09-07，
  
  录用：2026-09-12，
  
  纸质出版：2026-10-30
- 稿件说明：
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刘佳，玛依热·阿吉，谢耀慧，等.S1P/S1PR2信号轴通过阻断自噬流促进心肌成纤维细胞活化与胶原分泌而恢复自噬流可逆转纤维化［J］.陆军军医大学学报，2026，48（20）：2908-2919.DOI：10.16016/j.2097-0927.202608001.

Liu J，Aji M Y R，Xie Y H，et al.S1P/S1PR2 axis promotes cardiac fibroblast activation and collagen secretion by blocking autophagic flux， while restoring autophagic flux reverses fibrosis［J］.J Army Med Univ，2026，48（20）：2908-2919.DOI：10.16016/j.2097-0927.202608001.
刘佳，玛依热·阿吉，谢耀慧，等.S1P/S1PR2信号轴通过阻断自噬流促进心肌成纤维细胞活化与胶原分泌而恢复自噬流可逆转纤维化［J］.陆军军医大学学报，2026，48（20）：2908-2919.DOI：10.16016/j.2097-0927.202608001. DOI：

Liu J，Aji M Y R，Xie Y H，et al.S1P/S1PR2 axis promotes cardiac fibroblast activation and collagen secretion by blocking autophagic flux， while restoring autophagic flux reverses fibrosis［J］.J Army Med Univ，2026，48（20）：2908-2919.DOI：10.16016/j.2097-0927.202608001. DOI：

摘要

目的

心肌成纤维细胞（cardiac fibroblasts，CFs）异常活化及胶原过度沉积是心肌纤维化发生发展的关键病理环节。本研究旨在探讨1-磷酸鞘氨醇（sphingosine-1-phosphate，S1P）/1-磷酸鞘氨醇受体2（sphingosine-1-phosphate receptor 2，S1PR2）信号轴对CFs胶原分泌及纤维化表型的调控作用，并从自噬流角度阐明其分子机制。

方法

选取健康清洁级SD大鼠乳鼠3只（出生1~3 d，雌雄不限，体质量5~8 g），采用差速贴壁法分离培养大鼠原代CFs，通过波形蛋白（Vimentin）免疫荧光染色进行纯度鉴定。①探讨S1P/S1PR2信号轴对CFs活化与胶原分泌的影响，将CFs分为Control组、S1P组、S1P+S1PR2 特异性拮抗剂（JTE-013）组、S1P+小干扰RNA阴性对照（small interfering RNA negative control，si-NC）组及S1P+敲低S1PR2小干扰RNA（small interfering RNA targeting S1PR2，si-S1PR2）组，采用CCK-8法检测细胞活性，划痕实验检测细胞迁移能力，ELISA法检测细胞培养上清液中Ⅰ型胶原（collagen type Ⅰ，Col Ⅰ）及Ⅲ型胶原（collagen type Ⅲ，Col Ⅲ）含量，免疫荧光染色及Western blotting检测细胞中α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、S1PR2及鞘氨醇激酶1（sphingosine kinase 1，SphK1）的表达。②探究自噬流在S1P/S1PR2信号轴中的调控作用，将CFs分为Control组、S1P组、S1P+JTE-013 组、S1P+自噬诱导剂雷帕霉素（rapamycin，Rapa）组和S1P+自噬流阻断剂巴弗洛霉素A1（bafilomycin A1，Baf A1）组，采用mRFP-GFP-LC3双荧光腺病毒转染观察自噬流变化，Western blotting检测自噬相关蛋白［微管相关蛋白1轻链3（microtubule-associated protein 1 light chain 3，LC3）-Ⅱ/LC3-Ⅰ、p62、组织蛋白酶D（cathepsin D，Cath D）］及α-SMA的表达水平。

结果

原代培养的大鼠CFs生长状态良好且形态典型，经Vimentin免疫荧光鉴定阳性率达95%以上，纯度符合实验要求。①与Control组比较，S1P组CFs的活性与迁移能力显著增强，Col Ⅰ、Col Ⅲ分泌量显著增加，α-SMA荧光染色强度及α-SMA、S1PR2、SphK1蛋白表达显著上调（

0.05）。与S1P组比较，S1P+JTE-013组CFs的活性与迁移能力显著降低，Col Ⅰ、Col Ⅲ分泌量显著减少，α-SMA荧光染色强度及α-SMA、S1PR2、SphK1蛋白表达显著下调（

0.05）。与S1P+si-NC组比较，S1P+si-S1PR2组CFs的活性与迁移能力显著降低，Col Ⅰ、Col Ⅲ分泌量显著减少，α-SMA荧光染色强度及α-SMA、S1PR2、SphK1蛋白表达显著下调（

0.05）。②与Control组比较，S1P组细胞内黄色荧光斑点大量积聚，LC3-Ⅱ/LC3-Ⅰ比值及p62蛋白表达显著

上调，而Cath D蛋白表达显著下调（

0.05）。与S1P组比较，S1P+JTE-013组和S1P+Rapa组红色荧光斑点数显著增加，黄色斑点数显著减少，p62蛋白表达显著下调，Cath D蛋白表达显著上调（

0.05）。此外，与S1P组比较，S1P+JTE-013组中LC3-Ⅱ/LC3-Ⅰ比值显著降低，S1P+Rapa组中LC3-Ⅱ/LC3-Ⅰ比值显著升高（

0.05）；而与S1P组比较，S1P+Baf A1组自噬流进一步受阻，表现为红色斑点数显著减少、黄色斑点数显著增加，LC3-Ⅱ/LC3-Ⅰ比值显著升高，p62和α-SMA蛋白表达显著上调，Cath D蛋白表达显著下调，且Col Ⅰ、Col Ⅲ分泌量显著增加（

0.05）。

结论

S1P/S1PR2信号轴通过阻断自噬流降解，促进心肌成纤维细胞活化与胶原分泌，而恢复自噬流可有效逆转S1P诱导的CFs纤维化表型。

Abstract

Objective

Abnormal activation of cardiac fibroblasts (CFs) and excessive collagen deposition are critical pathological events in the development of myocardial fibrosis. This study aimed to investigate the regulatory role of the sphingosine-1-phosphate (S1P)/sphingosine-1-phosphate receptor 2 (S1PR2) signaling axis in collagen secretion and the fibrotic phenotype of CFs

and to elucidate the molecular mechanisms from the perspective of autophagic flux.

Methods

Three healthy clean-grade neonatal SD rats (aged 1 to 3 d

weighing 5 to 8 g

either sex) were selected

primary rat CFs were isolated and cultured using differential adhesion method

and their purity was confirmed by Vimentin immunofluorescence staining. ① To investigate the effects of the S1P/S1PR2 signaling axis on CF activation and collagen secretion

CFs were divided into Control group

S1P group

S1P+S1PR2 specific antagonist (JTE-013) group

S1P+small interfering RNA negative control transfection (si-NC) group

and S1P+S1PR2 knockdown (si-S1PR2) group. Cell viability and migration were detected using CCK-8 assay and scratch wound assay

and the contents of type Ⅰ collagen (Col Ⅰ) and type Ⅲ collagen (Col Ⅲ) in cell culture supernatant was measured with ELISA. The expression of α-smooth muscle actin (α-SMA)

S1PR2

and sphingosine kinase 1 (SphK1) was detected by immunofluorescence staining and Western blotting. ② To explore the regulatory role of autophagic flux in the S1P/S1PR2 signaling axis

CFs were divided into Control group

S1P group

S1P + JTE-013 group

S1P + autophagy inducer rapamycin (Rapa) group

and S1P + bafilomycin A1 (Baf A1

autophagic flux inhibitor) group. Changes in autophagic flux were observed using mRFP-GFP-LC3 dual-fluorescence adenovirus transfection

and the expression levels of autophagy-related proteins [microtubule-associated protein 1 light chain 3 (LC3-Ⅱ/LC3-Ⅰ)

p62

and cathepsin D (Cath D)] and α-SMA were detected by Western blotting.

Results

Primary cultured rat CFs exhibited robust growth with typical morphology

and the positive rate of Vimentin immunofluorescence identification was over 95%

meeting the experimental purity requirement. ① Compared with the Control group

the S1P group showed significantly enhanced CF proliferation and migration abilities

increased Col Ⅰ and Col Ⅲ secretion

enhanced α-SMA fluorescence staining intensity

and upregulated protein expression levels of α-SMA

S1PR2

and SphK1 (

0.05). Compared with the S1P group

CF viability and migration capacity were significantly decreased

Col Ⅰ and Col Ⅲ secretion was significantly reduced

and α-SMA fluorescence intensity along with α-SMA

S1PR2

and SphK1 protein expression were significantly decreased in the S1P+JTE-013 group (

0.05); similarly

compared with the S1P+si-NC group

all the aforementioned parameters were significantly reduced in the S1P+si-S1PR2 group (

0.05). ② Compared with the Control group

the S1P group exhibited significant accumulation of yellow fluorescent puncta

higher LC3-Ⅱ/LC3-Ⅰ ratio

and upregulated p62 expression

while downregulated Cath D expression (

0.05). Compared with the S1P group

the S1P+JTE-013 and S1P+Rapa groups showed significantly increased number of red fluorescent puncta

decreased number of yellow puncta

downregulated p62 and upregulated Cath D expression (

0.05). Additionally

compared with the S1P group

the LC3-Ⅱ/LC3-Ⅰ ratio was significantly decreased in the S1P+JTE-013 group

whereas increased in the S1P+Rapa group (

0.05). In contrast

compared with the S1P group

the S1P+Baf A1 group showed further blocked autophagic flux

manifested as significantly reduced number of red puncta and increased number of yellow puncta

increased LC3-Ⅱ/LC3-Ⅰ ratio

upregulated expression of p62 and α-SMA while downregulated Cath D

and enhanced secretion of Col Ⅰ and Col Ⅲ (

0.05).

Conclusion

The S1P/S1PR2 signaling axis promotes cardiac fibroblast activation and collagen secretion by blocking autophagic flux degradation

and restoring autophagic flux can effectively reverse the S1P-induced fibrotic phenotype in CFs.

关键词

Keywords

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