Department of Neurosurgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, PR China.
Henan Key Laboratory of Child Brain Injury, Institute of Neuroscience and Third Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan, PR China.
CNS Neurosci Ther. 2021 Jun;27(6):674-686. doi: 10.1111/cns.13626. Epub 2021 Feb 28.
Intracerebral hemorrhage (ICH) is a catastrophic cerebrovascular disease with high morbidity and mortality. Evidence demonstrated that sphingosine-1-phosphate receptor (S1PR) plays a vital role in inflammatory damage via the upregulation of CCL2 expression. However, whether S1PR3 is involved in blood-brain barrier (BBB) breakdown via CCL2 activation after ICH has not been described.
We investigated the expression profiles of all S1PRs using high-throughput RNA-seq analysis and RT-PCR. The potential role of S1PR3 and interaction between S1PR3 and CCL2 were evaluated via Western blotting, immunofluorescence, and flow cytometry. BBB disruption was examined via magnetic resonance imaging, transmission electron microscopy, and Evans blue extravasation. Microglial activation, proliferation, and polarization were assessed via histopathological analysis. The expression levels of CCL2, p-p38 MAPK, ICAM-1, and ZO-1 were examined in vitro and in vivo.
The present results showed that the levels of S1PR3 and its ligand, sphingosine 1-phosphate (S1P), were dramatically increased following ICH, which regulated the expression of CCL2 and p38MAPK. Moreover, reductions in brain edema volume, amelioration of BBB integrity, and improvements in behavioral deficits were achieved after the administration of CAY10444, an S1PR3 antagonist, to rats. Remarkably increased CCL2, p-p38MAPK, and ICAM-1 expression and decreased ZO-1 expression were observed in cocultured human astrocytes (HAs) and hCMEC/D3 cells after S1P stimulation. However, the expression levels of CCL2, p-p38 MAPK, and ICAM-1 were decreased and ZO-1 expression was increased after S1PR3 inhibition. In addition, microglial proliferation and M1 polarization were attenuated after CAY10444 administration.
To the best of our knowledge, this is the first demonstration of the neuroprotective role of S1PR3 modulation in maintaining BBB integrity by inhibiting the S1PR3-CCL2 axis after ICH, providing a novel treatment for ICH by targeting S1PR3.
脑出血（ICH）是一种发病率和死亡率都很高的灾难性脑血管病。有证据表明，鞘氨醇-1-磷酸受体（S1PR）通过上调 CCL2 的表达在炎症损伤中发挥重要作用。然而，ICH 后 S1PR3 是否通过 CCL2 激活参与血脑屏障（BBB）的破坏尚未描述。
我们使用高通量 RNA-seq 分析和 RT-PCR 研究了所有 S1PR 的表达谱。通过 Western blot、免疫荧光和流式细胞术评估了 S1PR3 的潜在作用以及 S1PR3 与 CCL2 之间的相互作用。通过磁共振成像、透射电子显微镜和 Evans 蓝渗出检测 BBB 破坏。通过组织病理学分析评估小胶质细胞的激活、增殖和极化。在体外和体内检测了 CCL2、p-p38 MAPK、ICAM-1 和 ZO-1 的表达水平。
本研究结果表明，ICH 后 S1PR3 及其配体鞘氨醇 1-磷酸（S1P）的水平显著升高，调节 CCL2 和 p38MAPK 的表达。此外，用 S1PR3 拮抗剂 CAY10444 处理大鼠后，脑水肿体积减少，BBB 完整性得到改善，行为缺陷得到改善。在 S1P 刺激后，共培养的人星形胶质细胞（HAs）和 hCMEC/D3 细胞中观察到 CCL2、p-p38MAPK 和 ICAM-1 表达显著增加，ZO-1 表达减少。然而，S1PR3 抑制后 CCL2、p-p38 MAPK 和 ICAM-1 的表达水平降低，ZO-1 的表达水平升高。此外，CAY10444 给药后，小胶质细胞增殖和 M1 极化减弱。
据我们所知，这是首次证明 S1PR3 调节通过抑制 ICH 后 S1PR3-CCL2 轴在维持 BBB 完整性中的神经保护作用，为 S1PR3 靶向治疗 ICH 提供了一种新的治疗方法。
