Background: Anti-podoplanin antibody (α-PDPN, clone 8.1.1) reduces microglia-mediated inflammation and decreases cerebral infarct volume in mice with stroke. However, the molecular mechanism by which this occurs is unknown. This study sought to systematically analyze the molecular mechanism of α-PDPN treatment on ischemia/reperfusion (I/R)-injured microglia.Methods: Microglia BV2 cells were pre-cultured with α-PDPN and then exposed to oxygen-glucose deprivation and reoxygenation (OGD-R) insult. The differentially expressed genes (DEGs) underwent a transcriptome sequencing technology analysis, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Quantitative real-time polymerase chain reaction (PCR) was performed to confirm the transcriptional expression of some DEGs. Results:The results showed that α-PDPN downregulated 338 genes and upregulated 340 genes in the BV2 cells. The GO items of the downregulated DEGs mainly involved biological processes, such as the response to the interferon (IFN), lipopolysaccharide-mediated signaling pathway, and the regulation of cell chemotaxis and migration. The upregulated molecular function mainly involved glucocorticoid-receptor binding. Further, the KEGG pathway analysis indicated that the enriched categories for the upregulated DEGs mainly involved the adenosine triphosphate (ATP) binding cassette transporters. However, the interleukin-17 signaling pathway, IFN signaling pathway, tumor necrosis factor signaling pathway, transforming growth factor beta (TGF-ꞵ) signaling pathway, nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathway, cytokine-cytokine receptor interaction, and chemokine signaling pathway were downregulated by the α-PDPN treatment.Conclusions: Numerous inflammation-related signaling pathways were regulated by the α-PDPN treatment in the OGD-R injured BV2 cells. This study provided further insights into the protective mechanism of α-PDPN treatment in ischemic stroke.

Background: Cerebral ischemia/reperfusion (I/R) injury involves the interaction between thrombosis and inflammatory pathways. The aim of this study was to explore the therapeutic effect of podoplanin neutralizing antibody (α-PDPN, clone 8.1.1) on I/R-induced thrombo-inflammation in a mouse model of ischemic stroke. Methods: Male C57BL/6 mice (weight: 22-25 g, aged 6-8 weeks, n=114) were subjected to transient middle cerebral artery occlusion (MCAO) and administered intracerebroventricular injection of α-PDPN (29 μg). Stroke outcomes and microvascular thromboses were examined by immunohistochemistry (IHC) and western blot analysis. In vitro, microglia BV2 cells were pre-treated with α-PDPN and then subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) insult. The microglia culture medium (MCM) was cocultured with vascular endothelial b.End3 cells. The MCM-induced bEnd.3 cells dysfunction were examined by western blot assays and IHC. Results: Blocking PDPN decreased the infarct size and ameliorated neurological deficit after MCAO without enhancing the risk of intracerebral hemorrhage. In addition, α-PDPN treatment significantly alleviated thrombus formation in the cerebral microvasculature. Furthermore, treatment with α-PDPN attenuated I/R-induced caspase-1 and gasdermin D expression in vivo and in vitro. The MCM containing α-PDPN reduced the expressions of von Willebrand factor and intercellular cell adhesion molecule-1 in bEnd.3 cells. Moreover, RNA sequencing analysis showed that α-PDPN decreased interferon signaling pathways in BV2 cells. Conclusions: Blocking PDPN can alleviate thrombo-inflammation in acute ischemic stroke by inhibiting caspase-1 expression in microglia, and indirectly reduce endothelial cell dysfunction. These data indicated the beneficial effects of blocking podoplanin during stroke in mice.