Background Abnormally activated neuroinflammation after traumatic brain injury is part of the main reasons for the poor prognosis. Astrocytes play a dual role in promoting repair or aggravating injury in the development of inflammation. Elongation factor Tu GTP binding domain containing protein (Eftud2) is an immunomodulator, which plays an important role in the occurrence and development of inflammation and tumor.
Objective To observe the effect of Eftud2 on the inflammatory response of astrocytes.
Methods Mice were randomly divided into sham operation group and traumatic brain injury group, and a traumatic brain injury model was constructed using a classical controlled cortical impact device. The contents of S100β and Eftud2 before and after injury were observed by immunofluorescence. The changes in motor function of mice before and after injury were evaluated by rotating rod and balance beam tests. Real-time fluorescence quantitative PCR was utilized to detect the changes of IL-1β, TNF-α, NLRP3 transcriptional level in tissues before and after injury. Eftud2 was knocked down in MA cells using smart interfering RNA and then stimulated to mimic inflammatory states in astrocyte lines (MA lines) using lipopolysaccharide. The transcriptional level changes of IL-6, IL-1β, TNF-α, GM-CSF, CXCL-10, IL-10 and myeloid differentiation factor 88 (MyD88) in MA cells were detected by real-time fluorescence quantitative PCR. The expression levels of Eftud2 and MyD88 in MA cells were detected by western blot.
Results In animal experiments, compared with the sham operation group, Eftud2 and S100β fluorescence intensity in the traumatic brain injury group increased (P < 0.01), and the transcription level of inflammatory factors IL-1β, TNF-α and NLRP3 around the injured tissue increased (P < 0.01). In cell experiments, Eftud2 protein expression level in MA cells was up-regulated after LPS stimulation (P < 0.05), and Eftud2 transcription level and fluorescence intensity of single cell was also increased (P < 0.01). Eftud2 protein expression level, transcription level and fluorescence intensity of single cell were decreased significantly in the knockout group (P < 0.01). After Eftud2 was knocked down in MA cells and stimulated by LPS, real-time fluorescence quantitative PCR results showed that the transcription level of IL-6 decreased (P < 0.05), and the transcription levels of IL-1β, TNF-α, GM-CSF, MyD88 and CXCL-10 also decreased (P < 0.01). Meanwhile, the transcription level of anti-inflammatory factor IL-10 was increased (P < 0.05). Western blot showed that the expression level of MyD88 protein was decreased (P < 0.05).
Conclusion Eftud2 may regulate the inflammatory response of astrocytes through a signaling pathway mediated by myeloid differentiation factor 88 (MyD88), and its mechanism needs to be further investigated.
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