目的

探讨创伤性脑损伤（TBI）后细胞焦亡相关基因的时序表达特征，构建关键分子竞争性内源性RNA（ceRNA）网络，为进一步研究TBI急性期神经炎症的干预靶点提供新视角。

方法

建立控制性皮层撞击（CCI）小鼠模型，设置假手术组及TBI后不同时间点的分组，包括损伤后0、1、2、3、4、6、12 h及1、3、7 d（每组3只），进行转录组测序。采用Mfuzz聚类分析探索焦亡相关基因转录水平在TBI后的时序表达特征，筛选TBI后12 h组和假手术组差异表达的信使RNA（mRNA）和长链非编码RNA（lncRNA）。基于生物信息学方法通过“miRDB”、“Starbase”、“LncBase v3.0”数据库构建ceRNA网络，并通过单细胞数据集、逆转录定量聚合酶链反应验证表达水平及双萤光素酶报告基因检测验证关键分子的直接结合。

结果

TBI后焦亡相关基因呈现2种表达模式，即分别在TBI后12 h和3 d达到表达高峰。TBI 12 h组和假手术组的差异分析筛选出533个差异mRNA（432个上调，101个下调）及103个差异lncRNA（51个上调，52个下调），其中Caspase-4、白介素（IL）-1α、IL-1β和IL-6为核心焦亡mRNA。构建的ceRNA网络包含4个lncRNA（F630028O10Rik、F730311O21Rik、BE692007和Mir17hg）、16个microRNA（mmu-miR-466a-3p等）及4个核心焦亡mRNA（Caspase-4、IL-1α、IL-1β和IL-6）。单细胞数据集分析证实IL-1β在TBI急性期高表达［log₂（FC）=3.072，P＜0.05］。双萤光素酶报告基因检测验证了F630028O10Rik/mmu-miR-466a-3p、mmumiR-466a-3p/IL-1β的直接结合，F630028O10Rik-WT和IL-1β-WT的相对萤光素酶活性分别下降了16.5%和27.1%（P＜0.05）。

结论

TBI后焦亡基因分为早期基因（12 h表达峰）和晚期基因（3 d表达峰），具有不同的功能。TBI后12 h焦亡相关的ceRNA网络显示IL-1β及其ceRNA调控轴（F630028O10Rik/miR-466a-3p/IL-1β）可能在TBI后急性期（12 h）的神经炎症中发挥核心作用，为TBI后急性期的干预靶点提供新的见解。

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.