The Neuroprotective Effect Of Nobiletin In Experimental Ischemic Stroke And Its Regulation On P-Akt, P-CREB, BDNF, Bcl-2and Claudin-5

Objectives:Ischemic stroke remains a leading cause of death and acquired disability worldwide in adults. Cerebral ischemia, induced by a reduction or complete blockade of blood flow to regions of the brain, is accompanied with an interdependent series of events including glutamate excitotoxicity, calcium overload, oxidative stress, nitric oxide production and inflammation, eventually leading to cell death. Despite the administration of intravenous thrombolysis is currently approved for acute ischemic stroke, its clinical application is still limited due to the narrow therapeutic window and safety concerns. Therefore, exploration of novel therapeutic agents becomes a major challenge in the field.The serine/threonine kinase Akt is a key downstream effector of the phosphoinositide-3-kinase pathway. The Akt activates and phosphorylates CREB at Ser133, resulting in the up-regulation of pro-survival CREB target genes such as BDNF and Bcl-2. Thus, Akt/CREB signaling pathway may be one propitious target for treatment of ischemic cerebral injury.NOB, a citrus polymethoxy flavonoid, is mainly extracted from a traditional herbal medicine pericarpium citri reticulatae and is abundantly present in the fruit pericarp of Citrus. In traditional Chinese medicine, dried citrus fruit peels are widely used as remedies to alleviate stomach upset, respiratory inflammatory syndromes and ringworm infections. NOB has attracted people’s attention due to its versatile health-promoting properties including anti-inflammatory, antioxidant and anticarcinogenic activities. Furthermore, NOB also displayed anti-atherogenic, anti-diabetic, hepatic-protective and neurotrophic effects. However, little is currently known regarding the exact role of NOB in ischemic stroke and the underlying mechanisms.This experiment successfully established the model of permanent middle cerebral artery occlusion in rats. Neurological deficit scores, infarct volume, and brain water content were evaluated at24h after stroke. Additionally, the activities of Akt, CREB, BDNF, Bcl-2and claudin-5in ischemic brain cortex were analyzed by the methods of immunohistochemistry, western blot and RT-qPCR. We here address two questions of whether NOB has neuroprotective effects on acute ischemia-induced neuronal death in cerebral cortex and what was the potential molecular mechanisms using a pMCAO model rat.Methods:Adult male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion (pMCAO) and randomly divided into five groups:Sham (sham-operated+0.05%Tween-80), MCAO (pMCAO+0.9%saline), Vehicle group (pMCAO+0.05%Tween-80), NOB-L (pMCAO+NOB10mg/kg) and NOB-H (pMCAO+NOB25mg/kg) groups. Rats were pre-administered intraperitoneally once daily for3days before surgery and then received once again immediately after surgery. At24h after MCAO, neurological deficit scores were evaluated by an examiner blinded to the experimental groups following a modified scoring system. Brain water content was measured using the standard wet-dry method, infarct volume was determined by2,3,5-triphenyltetrazolium chloride (TTC). Additionally, the activities of Akt, CREB, BDNF, Bcl-2and claudin-5in ischemic brain cortex were analyzed by the methods of immunohistochemistry, western blot and RT-qPCR.Results:1neurological score:Both MCAO and Vehicle group got higher scores than Sham group. Compared with Vehicle group, the neurological deficit scores were reduced in NOB-H group (Vehicle group vs. NOB-H group:3.28±0.177vs.2.17±0.232, P<0.05), but no significant difference was found between Vehicle group and NOB-L group (Vehicle group vs. NOB-L group: 3.28±0.177vs.2.78±0.222, P>0.05).2Brain water content measurement:Ipsilateral brain water content of Sham group was78.46±0.34%. Compared with Vehicle group, NOB-H group showed a decline in the percentage of brain water content (Vehicle group vs. NOB-H group:85.38±0.52%vs.83.38±0.41%,P<0.05). Though treatment with low doses of NOB reduced brain edema (NOB-L group:84.71±0.33%), it did not reach a significant level.3Brain infarction volume:No infarction was observed in the sham-operated group. In MCAO and Vehicle group, an extensive lesion was developed in both striatum and lateral cortex (Fig.1C). The infarct volume was significantly lessened from46.12±0.94%in Vehicle group to39.84±1.77%in the NOB-L group and36.07±2.32%in the NOB-H group (P<0.05, Fig. ID).4NOB’s effect on the expressions of p-Akt, p-CREB, BDNF, Bcl-2and claudin-5:High dose NOB significantly enhanced the expressions of p-Akt, BDNF, Bcl-2and claudin-5at protein levels(P<0.05). Both NOB-L and NOB-H group elevated the expressions of p-CREB compared with Vehicle group (P<0.05, Fig.3B), and unexpectedly NOB-L group got a higher level than NOB-H group. The mRNA expressions of Akt and BDNF were significantly induced by treatment with high doses of NOB (P<0.05, Fig.4A and B), but low doses of NOB did not display this effect. However, both NOB-L and NOB-H group elevated the gene levels of CREB and Bcl-2(P<0.05, Fig.4A and B).Conclusions:NOB protected the brain from damage caused by pMCAO; this effect may be associated with the activation of Akt/CREB signaling pathway and up-regulated expressions of BDNF, Bcl-2and claudin-5.