Inhibition of Connexin43 hemichannels with Gap19 protects cerebral ischemia/reperfusion injury via the JAK2/STAT3 pathway in mice

Abstract Functional disruption of the neurovascular unit may lead to aggravation of ischemic cerebral injury. Connexin43 (Cx43)-dependent gap junctional channels (GJCs) are critical in maintaining brain homeostasis. However, excessive opening of hemichannels (HCs) after cerebral ischemia may cause apoptosis and finally lead to amplification of ischemic injury. Previous studies indicated that Cx43 mimetic peptides Gap26 and Gap27 may protect cerebral ischemic injury, but the latest studies showed they also inhibit the opening of GJCs, which are beneficial for neuroprotection. Recent studies showed that Gap19 is a new specific inhibitor of Cx43 HCs. We investigated the role of Gap19 on cerebral ischemia/reperfusion (I/R) injury in a mouse model of middle cerebral artery occlusion (MCAO). Ventricle-injected Gap19 significantly alleviated infarct volume, neuronal cell damage and neurological deficits after ischemia, the neuroprotective effect of Gap19 was significant stronger than Gap26. Post-treatment with TAT-Gap19 still provided neuroprotection when it was administered intraperitoneally at 4 h after reperfusion. In addition, we found that Gap19 decreased the levels of cleaved caspase-3 and Bax and increased the level of Bcl-2, suggesting the anti-apoptotic activity of specifically blocking the Cx43 HCs. Furthermore, our data indicate that Gap19 treatment increased the levels of phosphorylated JAK2 and STAT3 both in vivo and in vitro. Gap19 inhibited hemichannel activity assessed by dye uptake in astrocytes. And we detected that pSTAT3 co-localized with Cx43 together in astrocytes after oxygen glucose deprivation (OGD) injury. Finally, AG490, a blocker of the JAK2/STAT3 pathway, could reverse the neuroprotective effects of Gap19 both in vivo and in vitro. Our experiment investigated the anti-apoptotic activity of Gap19, the specific inhibitor of Cx43 HCs, and the potential mechanisms. Our results demonstrated that Gap19 plays an anti-apoptotic role via activating the JAK2/STAT3 pathway after cerebral I/R injury, indicating that specific blocking of Cx43 HCs is a potential target for ischemic stroke.