Beneficial Effects Of Sulindac In Focal Cerebral Ischemia: A Positive Role In Wnt/β-catenin Pathway

Objective: Ischemic stroke with a high incidence is a leading cause ofdeath and permanent disability in adult worldwide. Accumulated evidenceshave established a critical role for inflammation in propagating neuronaldamage after cerebral ischemia. Sulindac, a cyclooxygenase inhibitor andnonsteroidal anti-inflammatory drug, is well known for its anti-inflammatoryproperties. However, the use of sulindac for the protection of acute cerebralischemia is still in an exploratory state. The mechanism of neuroprotection isalso needed to be further investigated. Wnt/β-Catenin signaling, whichoperates functions in gene transcription such as the claudin-5and Bcl-2, hasbeen identified as one pathway that regulates neuronal homeostasis duringdevelopment and adult life. Inhibition of the Wnt/β-catenin pathway has beenimplicated in the pathophysiology of neuronal death in cerebral ischemia. Itcan be suggested that pharmaceutical therapies, enhancing the activity ofWnt/β-catenin signaling pathway, will be beneficial in focal cerebral ischemia.This study was to investigate the potential neuroprotective effect of sulindacand its underlying mechanism in vivo, that whether it was correlated with theactivation of Wnt/β-catenin signaling pathway.Methods: Male, Sprague-Dawley rats underwent permanent middlecerebral artery ocllusion (pMCAO) as described by Longa previously. Thedrugs was administrated by oral according to different groups30minutesbefore cerebral ischemia. All the rats were randomly assigned to five groups:Sham group (sham-operation+0.01M PBS), Vehicle group (pMCAO+0.01M PBS) and Low (pMCAO+sulindac4mg/kg), Middle (pMCAO+sulindac10mg/kg) and High (pMCAO+sulindac20mg/kg) doses group. At24h after pMCAO, neurologic deficit scores were evaluated, brain watercontent was measured by wet-dry method and infarct size was analysised with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Meanwhile, mediatorsinvolved in Wnt/β-catenin signaling pathway including Dishevelled (Dvl),adenomatous poliosis coli (APC), P-β-catenin and β-catenin, as well as Bcl-2,Bax and claudin-5which could be regulated by the Wnt/β-catenin pathway,were determined with immunohistochemistry, reversetranscription-polymerase chain reaction and western blot.Results:1Comparied with Vehicle group, neurological deficit scores in Highgroup were significantly decreased at24h after pMCAO (P <0.05). Low andMiddle group also showed a decrease in neurological deficit scores, but therewere no significant differences as compared with Vehicle group (P>0.05).2In Sham group, the percentage of brain water content (BWC) was about79.20±0.73%. After pMCAO, the BWC of ipsilateral hemispheres was up to85.32±1.05%in Vehicle group. Both Middle and High doses of sulindacsignificantly decreased the BWC after ischemic stroke (Middle vs. Vehicle:84.20±0.62%vs.85.32±1.05%, P <0.05; High vs. Vehicle:83.07±1.22%vs.85.32±1.05%, P <0.01). However, there was no significant difference in thepercentage of BWC between Low and Vehicle group (Low vs. Vehicle:84.54±0.63%vs.85.32±1.05%, P>0.05).3Extensive lesion was developed in both striatum and lateral cortex inVehicle group. There was no difference in the infarct volume between Vehicleand Low group (P>0.05). However, the rats treated with High and Middledoses of sulindac showed a significant reduction in infarction size whencompared with Vehicle animals (P <0.05).4In Vehicle group, Dvl and β-catenin, which were two positiveregulators in Wnt/β-catenin pathway, were inhibited; as well as the expressionof APC and P-β-catenin which have a negative role in Wnt/β-catenin signalingpathway was upregulated at24h after cerebral ischemia. The high and middledoses of sulindac's treatment could restore the changes. However, there wasno significant difference in the four mediums expressions between Vehicle andLow group after pMCAO. 5Consistent with the previous studies, cerebral ischemia induced asignificant increase in apoptotic factor Bax and a significant decrease inanti-apoptotic factor Bcl-2. Compared with Vehicle treatment, Middle andHigh but not Low doses of sulindac significantly increased the expression ofBcl-2and decreased Bax expression at24h after pMCAO.6It was consistent with the previous studies that in Vehicle group,claudin-5was significantly down-regulated after pMCAO when comparedwith Sham group (P <0.05). Middle and High doses of sulindac significantlyup-regulated the claudin-5expression both at protein and mRNA levels.Compared with Vehicle group, Low group did not show significant increase inclaudin-5expression.Conclusions: sulindac improves functional outcome, decreases infarctsize and brain edema in cerebral ischemia. The neuroprotective effect ofsulindac is correlated with the activation of Wnt/β-catenin pathway, theinhibition of neuronal apoptosis and the upregulation of tight junction proteinin cerebral ischemia. The activation of Wnt/β-catenin signaling pathwaycontributes to the neuroprotection of sulindac in ischemic stroke.