Shao Qiang, Zhang Ying, Zhang Zhiyuan, Jiang Wei, Yin Yongcheng, Fang Yuepeng, Zhang Ce, Chen Qingfa, Ning Bin
Cheeloo College of Medicine, Jinan Central Hospital, Shandong University, Jinan, People's Republic of China.
Central Hospital Affiliated to Shandong First Medical University, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, Shandong, People's Republic of China.
J Inflamm Res. 2024 Oct 9;17:7123-7139. doi: 10.2147/JIR.S467940. eCollection 2024.
Post-spinal cord injury (SCI) inflammation correlates with neurologic recovery. Through sequencing, we explored the roles of a differentially expressed circRNA in mice after SCI, circGla, on inflammation and recovery of SCI.
The T8-T10 SCI model was established in C57BL6 mice. HE staining and RT-qPCR verified circGla upregulation results after injury obtained through sequencing. RNase R digestion and primer amplification experiments confirmed the circular properties of circGla. Nucleus and cytoplasm isolation experiments and FISH confirmed circGla expression in the astrocyte cytoplasm. AAV was used to establish a circGla knockdown mouse model. Behavioral tests were conducted to assess the recovery of the neurological function. The key inflammatory molecules after SCI were evaluated through MRI, RT-qPCR, and ELISA. For in vitro experiments, circGla was upregulated or knocked down in mouse astrocytes to detect its effect. The binding between miR-488 and circGla was confirmed through RIP and the dual luciferase experiment. RT-qPCR and ELISA confirmed the content correlation of the two molecules and the in vitro inflammatory function of miR-488. The binding experiment in astrocytes confirmed the binding between miR-488 and MEKK1 mRNA. Western blotting of MAPK pathway-related proteins confirmed that MEKK1 is a downstream effector for circGla and miR-488 in astrocytes.
Following SCI, the circular RNA circGla levels increased and it existed in the astrocyte cytoplasm. circGla knockdown reduced inflammation and improved neurological recovery in vivo. The correlation between circGla and proinflammatory factors was confirmed in vitro. circGla bound to miR-488, and the high miR-488 level was associated with the low astrocyte inflammatory state. miR-488 bound to MEKK1 mRNA, and upregulation or knockdown of circGla or miR-488 affected MAPK pathway-related protein expression.
Following SCI, downregulation of circGla expression in astrocytes can reduce inflammatory manifestations and stimulate long-term functional recovery in mice through miR-488 and MEKK1.
脊髓损伤（SCI）后的炎症反应与神经功能恢复相关。通过测序，我们探究了SCI后小鼠中差异表达的环状RNA circGla在SCI炎症及恢复过程中的作用。
在C57BL6小鼠中建立T8-T10节段的SCI模型。苏木精-伊红（HE）染色和逆转录定量聚合酶链反应（RT-qPCR）验证了测序所得的损伤后circGla上调结果。核糖核酸酶R消化和引物扩增实验证实了circGla的环状特性。细胞核与细胞质分离实验及荧光原位杂交（FISH）证实了circGla在星形胶质细胞细胞质中的表达。使用腺相关病毒（AAV）建立circGla敲低小鼠模型。进行行为测试以评估神经功能的恢复情况。通过磁共振成像（MRI）、RT-qPCR和酶联免疫吸附测定（ELISA）评估SCI后的关键炎症分子。在体外实验中，上调或敲低小鼠星形胶质细胞中的circGla以检测其作用。通过RNA免疫沉淀（RIP）和双荧光素酶实验证实了miR-488与circGla之间的结合。RT-qPCR和ELISA证实了这两种分子的含量相关性以及miR-488的体外炎症功能。星形胶质细胞中的结合实验证实了miR-488与丝裂原活化蛋白激酶激酶1（MEKK1）mRNA之间的结合。对丝裂原活化蛋白激酶（MAPK）通路相关蛋白进行蛋白质免疫印迹法检测，证实MEKK1是星形胶质细胞中circGla和miR-488的下游效应分子。
SCI后，环状RNA circGla水平升高，且存在于星形胶质细胞的细胞质中。circGla敲低可减轻体内炎症反应并改善神经功能恢复。体外实验证实了circGla与促炎因子之间的相关性。circGla与miR-488结合，且miR-488水平升高与星形胶质细胞低炎症状态相关。miR-488与MEKK1 mRNA结合，circGla或miR-488的上调或敲低会影响MAPK通路相关蛋白的表达。
SCI后，星形胶质细胞中circGla表达下调可通过miR-488和MEKK1减轻炎症表现并促进小鼠的长期功能恢复。
