Inhibition of Connexin 43 Hemichannels Alleviates Cerebral Ischemia/Reperfusion Injury via the TLR4 Signaling Pathway

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.

Targeting Connexin43 provides anti-inflammatory effects after intracerebral hemorrhage injury by regulating YAP signaling

“…However,Gap19 decreased the protein level but not the transcriptional activity of astrocytic Cx43 compared with that in the hemin group. Thus, Gap19 may affect the stability of Cx43 in astrocytes after ICH injury, which is in agreement with the effects of Gap19 on cerebral ischemia injury observed in our prior study [11].…”

“…Our data showed that TAT-Gap19 treatment did not in uence mouse neurobehavioral function under physiological conditions (P>0.05, Figure G-J), which is consistent with our previous studies [11,12]. The mNSSs of the mice were signi cantly increased by ICH damage, while TAT-Gap19 treatment markedly reduced mNSSs (11.20 ± 1.30 vs 6.50 ± 1.29, P<0.01, Figure 2G).…”

“…However, increasing evidence has demonstrated that blockers of Cx43Hcs can regulate many intracellular signaling pathways.Yin et al showed that carbenoxolone and Gap26, blockers of Cx43Hcs, signi cantly inhibit neuroin ammation in an astrocytic ischemia model by regulating PKC,PKB and Src signaling in both the membrane and cytoplasm [24].Li et al found that the Cx43 mimetic peptides Gap26 and Gap27 promote cytoplasmic degradation of astroglial Cx43 via the PI3K/Akt pathway in neonatal rats after ischemic brain injury [38]. Our previous study also found that Gap19 exerts its anti-in ammatory effects by inhibiting the activation of the JAK2/STAT3 and TLR4 signaling pathways in astrocytes after cerebral ischemic stroke [11,12]. Consistent with these results, we believe that modulating intracellular signaling pathways may change the function of astrocytic Cx43 on the cellular plasma membrane.…”

“…The Cx43 mimetic peptide Gap19 is used as a selective blocker of Cx43Hcs in the CNS under pathological conditions [20,21]. Our previous research revealed that Gap19 provides protective effects against cerebral ischemic stroke by reducing infarct volume and alleviating neurological de cits [11,12].…”

Meisoindigo Protects Against Focal Cerebral Ischemia-Reperfusion Injury by Inhibiting NLRP3 Inflammasome Activation and Regulating Microglia/Macrophage Polarization via TLR4/NF-κB Signaling Pathway

“…Cells were treated with LPS (1 µg/mL) and different concentrations (10, 30, 50, 100, and 150 mM) of meisoindigo at the beginning of OGD. The optimal dose of LPS was selected based on previous studies (Chen et al, 2018). Then the medium was removed, 10 µl of CCK-8 solution was subsequently added to each well.…”