SPP1/AnxA1/TIMP1 as Essential Genes Regulate the Inflammatory Response in the Acute Phase of Cerebral Ischemia-Reperfusion in Rats

Abstract

Background: Ischemic injury in stroke is followed by extensive neurovascular inflammation and changes in ischemic penumbra gene expression patterns. However, the key molecules involved in the inflammatory response during the acute phase of ischemic stroke remain unclear. Methods: Gene expression profiles of two rat ischemic stroke-related data sets, GSE61616 and GSE97537, were downloaded from the GEO database for Gene Set Enrichment Analysis (GSEA). Then, GEO2R was used to screen differentially expressed genes (DEGs). Furthermore, 170 differentially expressed intersection genes were screened and analyzed for Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Candidate genes and miRNAs were obtained by DAVID, Metascape, Cytoscape, STRING, and TargetScan. Finally, the rat middle cerebral artery occlusion-reperfusion (MCAO/R) model was constructed, and qRT-PCR was used to verify the predicted potential miRNA molecule and its target genes. Results: GO and KEGG analyses showed that 170 genes were highly associated with inflammatory cell activation and cytokine production. After cluster analysis, seven hub genes highly correlated with post-stroke neuroinflammation were obtained: Cxcl1, Kng1, Il6, AnxA1, TIMP1, SPP1, and Ccl6. The results of TargetScan further suggested that miR-340-5p may negatively regulate SPP1, AnxA1, and TIMP1 simultaneously. In the ischemic penumbra of rats 24 h after MCAO/R, the level of miR-340-5p significantly decreased compared with the control group, while the concentration of SPP1, AnxA1, and TIMP1 increased. Time-course studies demonstrated that the mRNA expression levels of SPP1, AnxA1, and TIMP1 fluctuated dramatically throughout the acute phase of cerebral ischemia-reperfusion (I/R). Conclusion: Our study suggests that differentially expressed genes SPP1, TIMP1, and ANXA1 may play a vital role in the inflammatory response during the acute phase of cerebral ischemia-reperfusion injury. These genes may be negatively regulated by miR-340-5p. Our results may provide new insights into the complex pathophysiological mechanisms of secondary inflammation after stroke.