S1P/S1PR2 axis promotes cardiac fibroblast activation and collagen secretion by blocking autophagic flux, while restoring autophagic flux reverses fibrosis

@article{jia2026s1ps1pr2,
  title = {{S1P/S1PR2 axis promotes cardiac fibroblast activation and collagen secretion by blocking autophagic flux, while restoring autophagic flux reverses fibrosis}},
  author = {Liu Jia and Wang Xumin},
  journal = {陆军军医大学学报},
  year = {2026},
  month = oct,
  doi = {10.16016/j.2097-0927.202608001},
  url = {https://doi.org/10.16016/j.2097-0927.202608001},
  note = {CC BY},
}

TY  - JOUR
TI  - S1P/S1PR2 axis promotes cardiac fibroblast activation and collagen secretion by blocking autophagic flux, while restoring autophagic flux reverses fibrosis
AU  - Liu Jia
AU  - Wang Xumin
PY  - 2026
DA  - 2026/10/30
JO  - 陆军军医大学学报
DO  - 10.16016/j.2097-0927.202608001
UR  - https://doi.org/10.16016/j.2097-0927.202608001
AB  - 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 (P<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 (P<0.05); similarly, compared with the S1P+si-NC group, all the aforementioned parameters were significantly reduced in the S1P+si-S1PR2 group (P<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 (P<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 (P<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 (P<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 (P<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.
SN  - 2097-0927
ER  -