Abstract:

Objective

To explore the temporal expression patterns of genes related to pyroptosis after traumatic brain injury （TBI） and construct a key molecular competitive endogenous RNA （ceRNA）network，providing a new perspective for further studying intervention targets of neuroinflammation during the acute phase of TBI.

Methods

A controlled cortical impact （CCI） mouse model was established，and groups were set up for sham surgery and different time points after TBI，including 0，1，2，3，4，6，12 h and 1，3，7 d post-injury （n=3 per group） for transcriptome sequencing. Mfuzz clustering analysis was used to explore the temporal expression characteristics of pyroptosis-related genes at the transcriptional level after TBI. Messenger RNA （mRNA） and long non-coding RNA （lncRNA） differentially expressed between TBI 12 h group and sham surgery group were screened. Based on bioinformatics methods，a competitive endogenous RNA （ceRNA） network was constructed using the“miRDB”，“Starbase”，and“LncBase v3.0” databases. The expression levels were verified through a single-cell dataset and RT-qPCR，and the direct binding of key molecules was validated using a dual-luciferase reporter assay.

Results

Pyroptosis-related genes exhibited two expression patterns after TBI，which reach their peak expression at 12 h and 3 d after TBI，respectively. Differential analysis between TBI 12 h group surgery and sham group identified 533 differentially expressed mRNA （432 upregulated and 101 downregulated） and 103 differentially expressed lncRNAs （51 upregulated and 52 downregulated），with Caspase-4，interleukin （IL）-1α，IL-1β，and IL-6 emerging as core pyroptosis mRNAs. The constructed ceRNA network comprised 4 lncRNAs （F630028O10Rik，F730311O21Rik，BE692007，and Mir17Hg），16 microRNAs （including mmumiR-466a-3p，etc.），and 4 core pyroptosis mRNAs （Caspase-4，IL-1α，IL-1β，and IL-6）. Single-cell dataset analysis confirmed the high expression of IL-1β during the acute phase of TBI. The dual-luciferase reporter gene assay verified the direct binding of F630028O10Rik/mmu-miR-466a-3p and mmu-miR-466a-3p/IL-1β. The relative luciferase activity of F630028O10Rik-WT and IL-1β-WT decreased by 16.5% and 27.1%，respectively （P＜0.05）.

Conclusion

The pyroptosis genes after TBI are divided into early genes （peak expression at 12 h） and late genes （peak expression at 3 d），which have different functions. A ceRNA network related to pyroptosis at 12 h after TBI was constructed，revealing that the IL-1β and its ceRNA regulatory axis （F630028O10Rik/miR-466a-3p/IL-1β） may play a central role in neuroinflammation during the acute phase （12 h） after TBI，providing new insights into intervention targets for the acute phase after TBI.

Key words: Traumatic brain injury, Neuroinflammation, Pyroptosis, Long non-coding RNA, Competing endogenous RNA