Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, 06032, USA.
Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, CT, 06032, USA.
Purinergic Signal. 2023 Sep;19(3):489-500. doi: 10.1007/s11302-023-09956-9. Epub 2023 Jul 13.
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.
鉴定新的潜在药物靶标蛋白及其治疗中风的可能机制至关重要。我们之前的研究表明，嘌呤能受体 P2X4（三磷酸腺苷的受体）的基因缺失和短期药理抑制可提供急性脑保护。然而，潜在的机制尚不清楚。因此，我们采用 RNA-Seq 技术来鉴定基因表达谱，并进行差异表达基因 (DEG) 的 qPCR 验证和通路分析。该分析确定了 DEG 在某些生物学过程中的作用，这些生物学过程负责 P2X4R 依赖性中风后的脑保护。我们使年轻和年老的雄性和雌性全局 P2X4 受体敲除 (P2X4RKO) 和同窝 WT (WT) 小鼠发生缺血性中风。三天后，处死小鼠，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 IPA 用于鉴定基因表达谱和生物途径。我们发现中风后 P2X4R KO 与 WT 组织之间有 2246 个 DEG。在这些 DEG 中，1920 个基因下调，325 个基因上调。GO/IPA 对前 300 个 DEG 的分析表明，炎症和细胞外基质成分基因富集。前 30 个 DEG 的 qPCR 验证显示，P2X4R KO 小鼠中有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 alpha (Ctla2a)，一种免疫抑制因子。这些数据表明，P2X4R 介导的中风后脑保护是通过在年轻和年老的雄性和雌性小鼠中减弱免疫调节途径来启动的。
