No abstract

Connexin 43 (Cx43) widely exists in all components of the neurovascular unit (NVU) and is a constituent of gap junctions and hemichannels. In physiological states, gap junctions are open for regular intercellular communication, and the hemichannels present low open probability in astrocytes. After cerebral ischemia, a large number of hemichannels are unusually opened, leading to cell swelling and even death. Most known hemichannel blockers also inhibit gap junctions and sequentially obstruct normal electrical cell-cell communication. In this study, we tested the hypothesis that Gap19, a selective Cx43-hemichannel inhibitor, exhibited neuroprotective effects on cerebral ischemia/reperfusion (I/R). An obvious improvement in neurological scores and infarct volume reduction were observed in Gap19-treated mice after brain ischemia induced by middle cerebral artery occlusion (MCAO). Gap19 treatment attenuated white matter damage. Moreover, Gap19 treatment suppressed the expression of Cx43 and Toll-like receptor 4 (TLR4) pathway-relevant proteins and prevented the overexpression of tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). To further explore downstream signaling, we established an in vitro model–oxygen glucose deprivation (OGD) to simulate ischemic conditions. Immunofluorescence staining showed that Cx43 co-existed with TLR4 in astrocytes. The hemichannel activity was increased after OGD and Gap19 could inhibit this effect on astrocytes. Gap19 substantially improved relative cell vitality and decreased the expression of Cx43, TLR4 and inflammatory cytokines in vitro. In addition, in the lipopolysaccharide (LPS) stimulation OGD model, Gap19 also exhibited a protective effect via inhibiting TLR4 pathway activation. In summary, our results showed that Gap19 exerted a neuroprotective effect after stroke via inhibition of the TLR4-mediated signaling pathway.

Background In the central nervous system (CNS), connexin 43 (Cx43) is mainly expressed in astrocytes and regulates astrocytic network homeostasis. Similar to Cx43 overexpression, abnormal excessive opening of Cx43 hemichannels (Cx43Hcs) on reactive astrocytes aggravates the inflammatory response and cell death in CNS pathologies. However, the role of excessive Cx43Hc opening in intracerebral hemorrhage (ICH) injury is not clear. Methods Hemin stimulation in primary cells and collagenase IV injection in C57BL/6J (B6) mice were used as ICH models in vitro and in vivo. After ICH injury, the Cx43 mimetic peptide Gap19 was used for treatment. Ethidium bromide (EtBr) uptake assays were used to measure the opening of Cx43Hcs. Western blotting and immunofluorescence were used to measure protein expression. qRT-PCR and ELISA were used to determine the levels of cytokines. Coimmunoprecipitation (Co-IP) and the Duolink in situ proximity ligation assay (PLA) were applied to measure the association between proteins. Results In this study, Cx43 expression upregulation and excessive Cx43Hc opening was observed in mice after ICH injury. Delayed treatment with Gap19 significantly alleviated hematoma volume and neurological deficits after ICH injury. In addition, Gap19 decreased inflammatory cytokine levels in the tissue surrounding the hematoma and decreased reactive astrogliosis after ICH injury in vitro and in vivo. Intriguingly, Cx43 transcriptional activity and expression in astrocytes were significantly increased after hemin stimulation in culture. However, Gap19 treatment downregulated astrocytic Cx43 expression through the ubiquitin-proteasome pathway without affecting Cx43 transcription. Additionally, our data showed that Gap19 increased Yes-associated protein (YAP) nuclear translocation. This subsequently upregulated SOCS1 and SOCS3 expression and then inhibited the TLR4-NFκB and JAK2-STAT3 pathways in hemin-stimulated astrocytes. Finally, the YAP inhibitor, verteporfin (VP), reversed the anti-inflammatory effect of Gap19 in vitro and almost completely blocked its protective effects in vivo after ICH injury. Conclusions This study provides new insight into potential treatment strategies for ICH injury involving astroglial Cx43 and Cx43Hcs. Suppression of abnormal astroglial Cx43 expression and Cx43Hc opening by Gap19 has anti-inflammatory and neuroprotective effects after ICH injury.