Neuroprotective effect of Apelin 13 on ischemic stroke by activating AMPK/GSK-3β/Nrf2 signaling

Abstract

BackgroundPrevious studies had showed that Apelin 13 could protect against apoptosis induced by ischemic/reperfusion (I/R). However, the mechanisms whereby Apelin 13 protected brain I/R remained to be elucidated. The present study was designed to determine whether Apelin 13 provided protection through AMPK/GSK-3β/Nrf2 pathway.MethodsIn vivo, the I/R model was induced and Apelin 13 was given intracerebroventricularly 15 min before reperfusion. The neurobehavioral scores, infarction volumes, and some cytokines in the brain were measured. For in vitro study, PC12 cells were used. To clarify the mechanisms, proteases inhibitors or siRNA were used. Protein levels were investigated by western blotting.ResultsThe results showed that Apelin 13 treatment significantly reduced infarct size, improved neurological outcomes, decreased brain edema, and inhibited cell apoptosis, oxidative stress, and neuroinflammation after I/R. Apelin 13 significantly increased the expression of Nrf2 and the phosphorylation levels of AMPK and GSK-3β. Furthermore, in cultured PC12 cells, the same protective effects were also observed. Silencing Nrf2 gene with its siRNA abolished the Apelin 13’s prevention of I/R-induced PC12 cell injury, oxidative stress, and inflammation. Inhibition of AMPK by its siRNA decreased the level of Apelin 13-induced Nrf2 expression and diminished the protective effects of Apelin 13. The interplay relationship between GSK-3β and Nrf2 was also verified with relative overexpression. Using selective inhibitors, we further identified the upstream of AMPK/GSK-3β/Nrf2 is AR/Gα/PLC/IP3/CaMKK.ConclusionsIn conclusion, the previous results showed that Apelin 13 protected against I/R-induced ROS-mediated inflammation and oxidative stress through activating the AMPK/GSK-3β pathway by AR/Gα/PLC/IP3/CaMKK signaling, and further upregulated the expression of Nrf2-regulated antioxidant enzymes.

Icariside II, a phosphodiesterase 5 inhibitor, attenuates cerebral ischaemia/reperfusion injury by inhibiting glycogen synthase kinase‐3β‐mediated activation of autophagy

“…There is mounting evidence that GSK‐3 plays an important role in regulating autophagic cell death. Phosphorylation of ser9 decreases GSK‐3β activity, whereas phosphorylation of tyr216 increases GSK‐3β activity (Duan et al, ; Peixoto, Nunes, & Garcia‐Osta, ). In the present study, icariside II robustly augmented GSK‐3β phosphorylation at Ser9 and reduced GSK‐3β phosphorylation at tyr216 in a dose‐dependent manner, suggesting that icariside II can inactivate GSK‐3β in neuronal cells, consistent with our previous study (Gao et al, ).…”

Attenuating effect of α-pinene on neurobehavioural deficit, oxidative damage and inflammatory response following focal ischaemic stroke in rat

“…After that, reperfusion started by withdrawing the filament. Successfully, the ischaemia was confirmed by an 80–90% reduction in cerebral blood flow (CBF) and was measured constantly up to 5 min by using a laser Doppler flowmeter (MBF3D; Moor Instruments, Devon, UK) …”

“…Dry weights (DW) of the brain samples were measured after 24 h at 120°C. The calculation of brain water content (BWC) was done by using following formula: [(WW − DW)/WW] × 100 …”

“…After neurobehavioural assessment, the animals were sacrificed under anaesthesia and the removal of brains was done quickly; they were sliced into 2‐mm thick coronal sections ( n = 5) for 15 min in saline at 4°C. The sections were treated by 2% TTC and incubated at 37°C for 15 min . The slices were then digitally photographed (Nikon).…”

“…Successfully, the ischaemia was confirmed by an 80-90% reduction in cerebral blood flow (CBF) and was measured constantly up to 5 min by using a laser Doppler flowmeter (MBF3D; Moor Instruments, Devon, UK). [19] Assessment of neurobehavioural function Following 24 h reperfusion, the neurological deficits were evaluated in rats by an observer who was blind to the rat groups. Neurological deficits were measured by using neurological severity scores (NSSs, Table 1) which was graded on a scale of 0-18 (0 as minimum and 18 as maximum).…”