Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.
Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.
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转录组分析揭示了新型的与年龄无关的免疫调节蛋白作为 P2X4 受体敲除小鼠缺血性中风后脑保护的一种模式。
DOI：
10.1007/s11302-023-09956-9
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发表时间：
2023
影响因子：
3.5
通讯作者：
Verma,Rajkumar
中科院分区：
文献类型：
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作者：
Gamiotea-Turro,Daylin;Cronin,ChunxiaC;Liang,BruceT;Verma,Rajkumar
迫切需要鉴定新的潜在药物靶蛋白及其治疗中风的合理机制。我们之前表明，P2X4（三磷酸腺苷（ATP）的嘌呤能受体）的基因缺失和短期药理学抑制可提供急性脑保护作用。然而，潜在的机制仍然未知。因此，我们采用RNA-Seq技术来鉴定基因表达谱和通路分析，然后对差异表达基因（DEG）进行qPCR验证。该分析确定了 DEG 在中风后 P2X4R 依赖性脑保护的某些生物过程中的作用。我们对年轻和年老的雄性和雌性全局 P2X4 受体敲除 (P2X4RKO) 小鼠和同窝 WT (WT) 小鼠进行缺血性中风。三天后，处死小鼠，使用 Trizol 分离总 RNA，并进行 RNA-Seq 和 NanoString 介导的 qPCR。 DESeq2、基因本体论 (GO) 和独创性路径分析 (IPA) 用于识别基因表达谱和生物学路径。我们发现中风后 P2X4R KO 与 WT 组织中存在 2246 个 DEG。在这些 DEG 中，P2X4R KO 中 1920 个基因下调，325 个基因上调。对前 300 个 DEG 的 GO/IPA 分析表明炎症和细胞外基质成分基因富集。对前 30 个 DEG 的 qPCR 验证显示 P2X4R KO 小鼠中两种常见的与年龄无关的基因下调：白细胞介素 6 (IL-6)（一种炎症细胞因子）和细胞毒性 T 淋巴细胞相关蛋白 2 α (Ctla2a)（一种免疫抑制因子）。这些数据表明，在年轻和老年小鼠中，中风后 P2X4R 介导的脑保护作用是通过免疫调节途径的减弱而启动的。
Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4, a purinergic receptor for adenosine triphosphate (ATP), provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-Seq technology to identify the gene expression profiles and pathway analysis followed by qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 receptor knock out (P2X4RKO) and littermate WT (WT) mice to ischemic stroke. After three days, mice were sacrificed, and total RNA was isolated using Trizol and subjected to RNA-Seq and NanoString-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify gene expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs. WT tissue after stroke. Out of these DEGs, 1920 genes were downregulated and 325 genes were upregulated in P2X4R KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (Il-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.