Earthworm extract improves cerebral ischemia–reperfusion injury through regulating microglia polarization and promoting cerebral angiogenesis in vitro and in vivo

Abstract

Promoting the polarization of microglia towards an anti-inflammatory M2 phenotype probably be exploited as a potential strategy for stroke. While earthworm has been shown to possess anti-inflammatory effects, its potential impact on microglial polarization and post-stroke recovery remains unclear. In in vitro experiments, BV2 mouse microglial cells were stimulated with lipopolysaccharide (LPS). Then, BV2 cells were given Earthworm extract (EWE). The expression levels of microglial phenotypic markers (CD16 and CD206), as well as cytokines (including IL-1β, VEGF, IL-10, and TGFβ), were assessed using techniques such as RT-PCR, immunofluorescence, ELISA, or Western blot. Subsequently, the viability and angiogenic potential of HCMEC/D3 human endothelial cells treated with conditioned media obtained from BV2 cells exposed to EWE (EWE-CM) were evaluated using migration assay, and tube formation assay. In in vivo experiments, C57 black mouse (C57 BL/6 mice) underwent middle cerebral artery occlusion and reperfusion (MCAO/R) and received daily tail injections of EWE for a duration of three days. The pathological condition of the brain was assessed using neurological deficit scores, 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin-eosin (H&E), and Nissl staining. The expression levels of CD206+/IBA-1+, CD16+/IBA-1+, and ki67+/Biotinylated LEL in the brain were assessed using immunofluorescence staining. The results demonstrated that EWE significantly suppressed pro-inflammatory cytokines and stimulated anti-inflammatory cytokines in LPS-induced microglia in vitro. Moreover, the EWE-CM enhanced the viability and tube-forming capacity of HCMEC/D3 cells, thereby promoting angiogenesis through activation of the Ang1/Tie2/Ang2 pathway. Furthermore, administration of EWE not only significantly ameliorated neurological deficits and reduced infarct volumes but also suppressed the activation of microglial cells in MCAO/R-induced mice in vivo. Thereby, EWE can modulate microglial cell M1/M2 polarization and enhance angiogenesis which is possibly activating the Ang1/Tie2/Ang2 pathway after cerebral ischemia, offering a potential therapeutic strategy for stroke.