Department of Pharmacy, Nanjing First Hospital, China Pharmaceutical University, Nanjing, China; Department of Pharmacy, Nanjing First Hospital, Nanjing Medical University, Nanjing, China.
Department of Pharmacy, Shanghai Children's Medical Center, Medical Department, Shanghai Jiao Tong University, Shanghai, China.
Int Immunopharmacol. 2023 May;118:110047. doi: 10.1016/j.intimp.2023.110047. Epub 2023 Mar 28.
Our previous studies have shown that berberine can improve the nerve function deficits in ischemic stroke by inhibiting inflammation. The cellular communication between astrocytes and neurons via exosomes might affect neurological function after ischemic stroke, which plays a vital role in the therapy of ischemic stroke.
The present study focused on the effects of exosomes released from astrocytes induced by the glucose and oxygen deprivation model with berberine pretreatment (BBR-exos) treatment for ischemic stroke and its regulatory mechanism.
Oxygen-glucose-deprivation/Reoxygenation (OGD/R)-treated primary cells were used to mimic cerebral ischemia/reperfusion conditions in vitro. With the treatment of BBR-exos and exosomes released from primary astrocytes induced by the glucose and oxygen deprivation model (OGD/R-exos), the cell viability was detected. C57BL/6J mice were used to establish middle cerebral artery occlusion/reperfusion (MCAO/R) model. The anti-neuroinflammation effects of BBR-exos and OGD/R-exos were evaluated. Subsequently, the key miRNA in BBR-exos was identified by exosomal miRNA sequencing and cell validation. miR-182-5p mimic and inhibitors were provided to verify the effects in inflammation. Finally, the binding sites between miR-182-5p and Rac1 were predicted online and verified by using a dual-luciferase reporter assay.
BBR-exos and OGD/R-exos both improved the decreased activity of OGD/R-induced neurons, and decreased the expression of IL-1β, IL-6 and TNF-α (all P ＜ 0.05), which reduced neuronal injury and inhibited neuroinflammation in Vitro. And BBR-exos showed better effects (P ＜ 0.05). The same effect has been verified in vivo experiments: BBR-exos and OGD/R-exos both reduced cerebral ischemic injury and inhibited neuroinflammation in MCAO/R mice (all P ＜ 0.05). Likewise, BBR-exos showed better effects (P ＜ 0.05). The exosomal miRNA sequencing results showed that miR-182-5p was highly expressed in BBR-exos and inhibited neuroinflammation by targeting Rac1 (P ＜ 0.05).
BBR-exos can carry miR-182-5p to injured neurons and inhibit the expression of Rac1, which could inhibit neuroinflammation and improved brain injury after ischemic stroke.
我们之前的研究表明，小檗碱可以通过抑制炎症来改善缺血性中风的神经功能缺损。星形胶质细胞和神经元之间通过外泌体的细胞通讯可能会影响缺血性中风后的神经功能，这在外泌体治疗缺血性中风中起着至关重要的作用。
本研究重点研究了小檗碱预处理后星形胶质细胞释放的外泌体（BBR-exos）对缺血性中风的作用及其调节机制。
用氧葡萄糖剥夺/再氧合（OGD/R）处理的原代细胞在体外模拟脑缺血再灌注条件。用 BBR-exos 和葡萄糖和氧剥夺模型诱导的原代星形胶质细胞释放的外泌体（OGD/R-exos）处理，检测细胞活力。用 C57BL/6J 小鼠建立大脑中动脉闭塞/再灌注（MCAO/R）模型。评价 BBR-exos 和 OGD/R-exos 的抗神经炎症作用。随后，通过外泌体 miRNA 测序和细胞验证鉴定 BBR-exos 中的关键 miRNA。提供 miR-182-5p 模拟物和抑制剂来验证炎症中的作用。最后，通过双荧光素酶报告基因检测在线预测 miR-182-5p 和 Rac1 之间的结合位点，并进行验证。
BBR-exos 和 OGD/R-exos 均改善了 OGD/R 诱导的神经元活性降低，并降低了 IL-1β、IL-6 和 TNF-α 的表达（均 P ＜0.05），从而减少了体外神经元损伤和抑制了神经炎症。而且 BBR-exos 表现出更好的效果（P ＜0.05）。体内实验也验证了相同的效果：BBR-exos 和 OGD/R-exos 均减轻了 MCAO/R 小鼠的脑缺血损伤并抑制了神经炎症（均 P ＜0.05）。同样，BBR-exos 表现出更好的效果（P ＜0.05）。外泌体 miRNA 测序结果显示，miR-182-5p 在 BBR-exos 中高表达，通过靶向 Rac1 抑制神经炎症（P ＜0.05）。
BBR-exos 可以携带 miR-182-5p 到受损神经元，并抑制 Rac1 的表达，从而抑制缺血性中风后的神经炎症和改善脑损伤。
