TGN-020 Alleviate Inflammation and Apoptosis After Cerebral Ischemia-Reperfusion Injury in Mice Through Glymphatic and ERK1/2 Signaling Pathway

Post-stroke acute inhibition of Aquaporin 4 (AQP4) is known to exacerbate inflammation and Apoptosis, yet the underlying mechanisms are not fully understood. The objective of this study was to investigate the specific mechanism of inflammation and Apoptosis following cerebral ischemia-reperfusion (I/R) injury using the AQP4-specific inhibitor, N-(1,3,4-thiadiazol-2-yl) pyridine-3-carboxamide dihydrochloride (TGN-020). Ischemic stroke was induced in mice using the middle cerebral artery occlusion (MCAO) model. The C57/BL6 mice were randomly divided into three groups as follows: sham operation, I/R 48 h, and TGN-020 + I/R 48 h treatment. All mice were subjected to a series of procedures. These procedures encompassed 2,3,5-triphenyltetrazolium chloride (TTC) staining, neurological scoring, fluorescence tracing, western blotting, immunofluorescence staining, and RNA sequencing (RNA-seq). The glymphatic function in the cortex surrounding cerebral infarction was determined using tracer, glial fibrillary acid protein (GFAP), AQP4 co-staining, and beta-amyloid precursor protein (APP) staining; differential genes were detected using RNA-seq. The influence of TGN-020 on the extracellular signal-regulated kinase 1/2 (ERK) 1/2 pathway was confirmed using the ERK1/2 pathway agonists Ro 67-7467. Additionally, we examined the expression of inflammation associated with microglia and astrocytes after TGN-020 and Ro 67-7467 treatment. Compared with I/R group, TGN-020 alleviated glymphatic dysfunction by inhibiting astrocyte proliferation and reducing tracer accumulation in the peri-infarct area. RNA-seq showed that the differentially expressed genes were mainly involved in the activation of astrocytes and microglia and in the ERK1/2 pathway. Western blot and immunofluorescence further verified the expression of associated inflammation. The inflammation and cell Apoptosis induced by I/R are mitigated by TGN-020. This mitigation occurs through the improvement of glymphatic function and the inhibition of the ERK1/2 pathway.

Glymphatic system; Inflammation; Ischemic stroke; RNA-seq; TGN-020.