Fig 2: Schematic showing potential roles of astrocytic Cx43, hemichannels, and GJIC during OGD/R injury. Under normal conditions, astrocytic Cx43 is expressed in the plasma membrane and assembled into hemichannels that are normally closed. Hemichannel-hemichannel interactions induce the formation of GJIC between adjacent astrocytes, which permits the exchange of ions and small molecules; also, plasma membrane’s Cx43 was phosphorylated at Ser368 site. In such circumstances, astrocytes, together with those resting microglia, function as a supportive assistant for healthy neurons. OGD/R injury caused abnormal hemichannel opening and consequent substantial astrocytic ATP release. It also induced microglial activation with a predominance of the pro-inflammatory cytokine-releasing M1 subtype. Extracellular ATP induced further microglial activation and pro-inflammatory cytokine release, and these pro-inflammatory cytokines induced further opening of astrocytic hemichannels. SalB reversed these effects and thus provided protection against OGD/R injury. This suggests the existence of a vicious cycle in which astrocytic hemichannel opening and pro-inflammatory microglial activation reinforce each other following OGD/R injury. This vicious cycle may account for secondary injury and extended damage after OGD/R injury; OGD/R injury caused gap junction internalization, which may account for the astrocytic uncoupling events. It also decreased plasma membrane levels of Ser368-phosphorylated Cx43 while increasing plasma membrane levels of Ser373-phosphorylated Cx43, Ser265-phosphorylated Cx43, and Src’s Tyr416-phosphorylated activated form. The activated Src may well have phosphorylated Cx43 at Tyr265 and further induced gap junction internalization or autophagy. SalB directly inhibits Src, which may allow it to exert protective effects by attenuating Cx43 internalization. CBX, a non-selective hemichannel and GJIC inhibitor, did not apparently affect Cx43 phosphorylation, but it inhibited PKC and Src activity

Fig 3: Effects of Vinp on the expression of Cx43, p-Cx43, AKT, and p-AKT in astrocytes after OGD/R. (A) Representative western blot images of Cx43 and p-Cx43, and quantification of Cx43, p-Cx43, and p-Cx43/Cx43. (B) Representative western blot images of AKT and p-AKT, and quantification of AKT, p-AKT and p-AKT/AKT. Data are shown as the mean ± SEM (n = 3 in each group). *P < 0.05, **P < 0.01, ***P < 0.001 using one-way ANOVA followed by Tukey’s post hoc tests. Vinp: Vinpocetine; LY: LY294002; OGD/R: oxygen-glucose deprivation/reoxygenation; BKM: BKM120.

Fig 4: OGD/R injury with SalB or CBX application influenced astrocytic phosphorylated Cx43 and corresponding kinases. a1, a2 OGD/R injury had no significant effect on cytoplasmic PKCe levels but significantly increased plasma membrane levels of PKCe and its Ser729-phosphorylated activated state. SalB and CBX reduced PKCe levels in the plasma membrane and increased them in the cytoplasm. Conversely, the OGD/R group’s Ser368-phosphorylated Cx43 levels were decreased in the plasma membrane and increased in the cytoplasm. SalB reversed these effects, but CBX reduced Ser368-phosphorylated Cx43 levels. b1, b2 The OGD/R group exhibited decreased cytoplasmic levels of Thr308-phosphorylated PKB, though SalB reversed this effect. There were no significant changes in the plasma membrane levels. Furthermore, the OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Ser373-phosphorylated Cx43. SalB reduced the plasma membrane levels but further increased the cytoplasmic levels. CBX had little effect on the levels of Ser373-phosphorylated Cx43 and Thr308-phosphorylated PKB. c1, c2 The OGD/R group exhibited elevated cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. The OGD/R group also exhibited elevated plasma membrane levels of Src, which may be related to the elevated Tyr416-phosphorylated Src levels. SalB increased the plasma membrane levels of Src’s Tyr527-phosphorylated deactivated form but did not significantly affect plasma membrane levels of Tyr416-phosphorylated Src. CBX significantly reduced cytoplasmic and plasma membrane levels of Tyr416-phosphorylated Src. Furthermore, the OGD/R group exhibited increased cytoplasmic and plasma membrane levels of Tyr265-phosphorylated Cx43. SalB reversed this effect, but CBX achieved only non-significant reduction of the plasma membrane levels. We evaluated the statistical significance with ANOVA and Duncan’s multiple comparisons test. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig 5: Redistribution of astrocytic Cx43 after OGD/R injury and the effects of SalB and CBX. We cultured primary astrocytes and performed cytoimmunofluorescent staining for Cx43. a1 In the normal group, Cx43 was mainly expressed discontinuously in the plasma membrane. b1 At high magnification, Cx43 mainly expressed at the gap junction and some were punctate distributed. a2, b2 In the OGD/R group, Cx43 was mainly expressed in the cytoplasm, which existed in the shape of block and grain. a3, b3 Compared to the OGD/R group, the OGD/R-SalB group exhibited weaker cytoplasmic Cx43 staining but enhanced plasma membrane Cx43 staining. The Cx43 expressed at gap junctions was morphologically similar to that in the normal group. a4, b4 The OGD/R-CBX group exhibited staining results similar to those of the OGD/R-SalB group, though the Cx43 at gap junctions covered a larger area than in the control and OGD/R-SalB groups. Scale bar = 50 µm