Vinpocetine Protects Against Cerebral Ischemia-Reperfusion Injury by Targeting Astrocytic Connexin43 via the PI3K/AKT Signaling Pathway

What is the EXACT METHOD your doctor is using to stop reperfusion injury? NOTHING? Then what research is your doctor working on to solve that problem?  You just might want to save those neurons that are dying during reperfusion. I'd suggest charging your doctor $1000 a dead neuron, that might concentrate the mind. Nothing else seems to get survivors closer to 100% recovery.

• reperfusion injury (26)

 

Vinpocetine Protects Against Cerebral Ischemia-Reperfusion Injury by Targeting Astrocytic Connexin43 via the PI3K/AKT Signaling Pathway

Mingming Zhao¹, Shuai Hou¹, Liangshu Feng¹, Pingping Shen¹, Di Nan¹, Yunhai Zhang^2,3, Famin Wang^2,3, Di Ma^1* and Jiachun Feng^1*

• ¹Department of Neurology and Neuroscience Center, The First Hospital of Jilin University, Changchun, China
• ²Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, China
• ³Jiangsu Key Laboratory of Medical Optics, Suzhou, China

Vinpocetine (Vinp) is known for its neuroprotective properties. However, the protective mechanism of Vinp against cerebral ischemia/reperfusion (I/R) injury should be further explored. This study was designed to investigate the neuroprotective effects of Vinp against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro and cerebral I/R injury in vivo and explore whether this mechanism would involve enhancement of astrocytic connexin 43 (Cx43) expression via the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. In vitro, we detected astrocytic viability and extracellular nitric oxide by an assay kit, intracellular reactive oxygen species by a DCFH-DA probe, inflammation and apoptosis-related protein expression by immunofluorescence staining, and the astrocytic apoptosis rate by flow cytometry. In vivo, we measured the cerebral infarction volume, superoxide dismutase activity, malondialdehyde content, and the expression of inflammation and apoptosis-related proteins. The results indicated that Vinp ameliorated the detrimental outcome of I/R injury. Vinp attenuated astrocytic injury induced by OGD/R and reduced cerebral infarction volume and cerebral edema in rats with cerebral I/R injury. Moreover, Vinp reduced oxidative stress, inflammation, and apoptosis induced by cerebral I/R injury in brain tissues. Meanwhile, Vinp increased p-Cx43 and p-AKT expression, and the p-Cx43/Cx43 and p-AKT/AKT ratio, which was decreased by cerebral I/R injury. Coadministration of PI3K inhibitors LY294002 and BKM120 blunted the effects of Vinp. This study suggests that Vinp protects against cerebral I/R injury via Cx43 phosphorylation by activating the PI3K/AKT pathway.

Introduction

Ischemic stroke has high morbidity and mortality and seriously affects patient quality of life (Ribeiro et al., 2015). Timely recovery of blood and oxygen supply to the ischemic brain tissue is essential for ischemic penumbra survival. Thrombolytic therapy is the best treatment option for ischemic stroke (Sheth et al., 2015). However, reperfusion aggravates the damage and provokes dysfunction through a cascade of events such as calcium overload, excitotoxicity, oxidative stress, inflammatory responses, and apoptosis, which are collectively termed “ischemia-reperfusion injury” (I/R injury) (Dirnagl et al., 1999). Therefore, effectively blocking the cascade of cerebral I/R injury and exploring effective drugs for the treatment of ischemic stroke are very important.

Astrocytes are abundant in the central nervous system, and they play essential roles in maintaining brain function under physiologic conditions and in influencing neuronal survival under pathologic conditions, such as cerebral I/R injury and other brain insults (Garman, 2011; Falkowska et al., 2015; Verkhratsky et al., 2017). During ischemic stroke, astrocytes may be activated and produce and release reactive oxygen species (ROS), pro-inflammatory cytokines, and other factors that may negatively influence the survival of neurons in the penumbra (Swanson et al., 2004). Thus, preventing astrocytic inflammatory and apoptotic effects may be a promising strategy for neuroprotection in ischemic stroke (Cekanaviciute and Buckwalter, 2016; Choudhury and Ding, 2016; Liu and Chopp, 2016).

The PI3K/AKT signaling pathway regulates a wide range of cellular functions, including cellular differentiation, proliferation, inflammation, and apoptosis (Cantley, 2002). Studies have shown that phosphorylation of AKT (Ser473) reduces neuronal apoptosis caused by cerebral I/R injury (Fukunaga and Kawano, 2003; Zhao et al., 2006), and LY294002-mediated inhibition of the PI3K/AKT pathway blocked the cardioprotective effect of atorvastatin against I/R injury in cardiocytes by downregulating Connexin 43 (Cx43) (Bian et al., 2015). Moreover, activated AKT can phosphorylate the C-terminal Ser373 residue of Cx43 (Solan and Lampe, 2014). Since Cx43 is the most commonly expressed gap junction protein in astrocytes (Orellana et al., 2011), and increased Cx43 expression can reduce neuronal damage after cerebral I/R (Nakase et al., 2003), we speculate that Cx43 is involved in the PI3K/AKT pathway’s protective effects against cerebral I/R injury.

Vinpocetine (Vinp) is a semi-synthetic alkaloid derivative isolated from the leaves of Phyllostachys pubescens. Its anti-inflammatory and anti-platelet aggregation effects on improving cerebral blood flow, brain metabolism, and cognition have been confirmed by various studies (Zhang et al., 2018; Zhang et al., 2018). Vinp has been widely used in the treatment of stroke, cerebral arteriosclerosis, and chronic cerebral insufficiency, and it exhibits unique advantages in the treatment of dementia and epilepsy. A previous study showed that Vinp similarly decreased the inflammatory response by inhibiting NF-κB and TNF-α expression after cerebral I/R injury (Wang et al., 2014); however, its specific mechanism remains unknown. Cerebral I/R injury can activate both astrocytes and microglia, which may produce inflammatory cytokines and other toxic mediators (Kim et al., 2016; Duris et al., 2018). Microglial TLR4/MyD88/NF-κB has been shown to be one of the mechanisms by which Vinp protects against cerebral I/R injury (Wu et al., 2017). However, so far, no study has focused on whether Vinp’s protective effects against cerebral I/R injury is related to astrocytes. Hence, we hypothesized that Vinp may affect astrocytic Cx43 via the PI3K/AKT pathway and thereby provide neuroprotection.

In this study, we explored the neuroprotective roles of vinpocetine against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro and cerebral I/R injury in vivo and explore whether this mechanism would involve enhancement of astrocytic connexin 43 (Cx43) expression via the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway.