Department of Cerebrovascular Surgery, Neurosurgery Center, Engineering Technology Research Center of Education Ministry of China on Diagnosis and Treatment of Cerebrovascular Disease, Zhujiang Hospital, Southern Medical University, Guangzhou, 510280, Guangdong, China.
Department of Physiology and Pharmacology, Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, CA, 92354, USA.
J Neuroinflammation. 2024 Jul 21;21(1):178. doi: 10.1186/s12974-024-03171-y.
Reactive astrocytes participate in various pathophysiology after subarachnoid hemorrhage (SAH), including neuroinflammation, glymphatic-lymphatic system dysfunction, brain edema, BBB disruption, and cell death. Astrocytes transform into two new reactive phenotypes with changed morphology, altered gene expression, and secretion profiles, termed detrimental A1 and beneficial A2. This study investigates the effect of 67LR activation by PEDF-34, a PEDF peptide, on neuroinflammation and astrocyte polarization after the experimental SAH.
A total of 318 male adult Sprague-Dawley rats were used in experiments in vivo, of which 272 rats were subjected to the endovascular perforation model of SAH and 46 rats underwent sham surgery. 67LR agonist (PEDF-34) was administrated intranasally 1 h after SAH. 67LR-specific inhibitor (NSC-47924) and STAT1 transcriptional activator (2-NP) were injected intracerebroventricularly 48 h before SAH. Short- and long-term neurological tests, brain water content, immunostaining, Nissl staining, western blot, and ELISA assay were performed. In experiments in vitro, primary astrocyte culture with hemoglobin (Hb) stimulation was used to mimic SAH. The expression of the PEDF-34/67LR signaling pathway and neuro-inflammatory cytokines were assessed using Western blot, ELISA, and immunohistochemistry assays both in vivo and in vitro.
Endogenous PEDF and 67LR expressions were significantly reduced at 6 h after SAH. 67LR was expressed in astrocytes and neurons. Intranasal administration of PEDF-34 significantly reduced brain water content, pro-inflammatory cytokines, and short-term and long-term neurological deficits after SAH. The ratio of p-JNK/JNK and p-STAT1/STAT1 and the expression of CFB and C3 (A1 astrocytes marker), significantly decreased after PEDF-34 treatment, along with fewer expression of TNF-α and IL-1β at 24 h after SAH. However, 2-NP (STAT1 transcriptional activator) and NSC-47924 (67LR inhibitor) reversed the protective effects of PEDF-34 in vivo and in vitro by promoting A1 astrocyte polarization with increased inflammatory cytokines.
PEDF-34 activated 67LR, attenuating neuroinflammation and inhibiting astrocyte A1 polarization partly via the JNK/STAT1 pathway, suggesting that PEDF-34 might be a potential treatment for SAH patients.
反应性星形胶质细胞参与蛛网膜下腔出血（SAH）后的多种病理生理学过程，包括神经炎症、糖液-淋巴管系统功能障碍、脑水肿、血脑屏障破坏和细胞死亡。星形胶质细胞转变为两种具有改变形态、改变基因表达和分泌谱的新的反应性表型，分别称为有害的 A1 和有益的 A2。本研究探讨了 PEDF 肽 PEDF-34 激活 67LR 对实验性 SAH 后神经炎症和星形胶质细胞极化的影响。
共 318 只成年雄性 Sprague-Dawley 大鼠用于体内实验，其中 272 只大鼠接受血管内穿剌法 SAH 模型，46 只大鼠接受假手术。SAH 后 1 小时经鼻内给予 67LR 激动剂（PEDF-34）。SAH 前 48 小时，经脑室注射 67LR 特异性抑制剂（NSC-47924）和 STAT1 转录激活剂（2-NP）。进行短期和长期神经功能测试、脑水含量测定、免疫染色、尼氏染色、Western blot 和 ELISA 检测。在体外实验中，使用血红蛋白（Hb）刺激原代星形胶质细胞培养来模拟 SAH。通过 Western blot、ELISA 和免疫组织化学检测体内和体外 PEDF-34/67LR 信号通路和神经炎症细胞因子的表达。
SAH 后 6 小时，内源性 PEDF 和 67LR 的表达明显降低。67LR 表达于星形胶质细胞和神经元中。鼻内给予 PEDF-34 可显著降低 SAH 后脑水含量、促炎细胞因子和短期及长期神经功能缺损。PEDF-34 处理后，p-JNK/JNK 和 p-STAT1/STAT1 比值以及 CFB 和 C3（A1 星形胶质细胞标志物）的表达均降低，SAH 后 24 小时 TNF-α和 IL-1β的表达减少。然而，2-NP（STAT1 转录激活剂）和 NSC-47924（67LR 抑制剂）通过促进 A1 星形胶质细胞极化和增加炎症细胞因子，逆转了 PEDF-34 在体内和体外的保护作用。
PEDF-34 激活 67LR，减轻神经炎症，抑制星形胶质细胞 A1 极化，部分通过 JNK/STAT1 通路，提示 PEDF-34 可能是 SAH 患者的一种潜在治疗方法。
