Neuroprotective Effects Of Paeoniflorin Against Middle Cerebral Artery Occlusion Injury In Rats And Its Mechanism

Background and objective: Ischemic stroke is a common disease,which has a high prevalence, annually increasing incidence and morbidity,ranking as the third major cause of death after myocardial infarction andcancer in our country. Development of drugs to treat cerebral ischemicstroke is one of the hot spots of medicine and academia nowadays. Peony isa kind of commonly traditional Chinese medicine, which can lowerabdominal cramps, relieve pain and improve blood circulation. One of itsmain active ingredient is paeoniflorin which is potentially effective inspasmolysis, anticoagulation and anti-inflammatory. Recent studies haveshown that paeoniflorin could significantly improve neurological scoresand reduce the infarction volume in rats induced by cerebral ischemia andreperfusion injury. However, there is no unified understanding of theoptimal administration method of paeoniflorin. Besides, the mechanisms ofits neuroprotection haven’t been under way. It’s the aim that to investigatethe neuroprotective effects and its mechanism of paeoniflorin on cerebralischemia-reperfusion injury in rats and to provide new theoretical basis for clinical application of paeoniflorin through the establishment of middlecerebral artery occlusion (MCAO)model in this study.The first part: Investigation of the optimal administration methodof paeoniflorinMethods: A total of72♂SD rats were randomly divided into6groups:Sham operation group(Sham), cerebral ischemia-reperfusion modelgroup（I/R）and different concentration of paeoniflorin groups. The middlecerebral artery occlusion in rats were made by inserting a modifiedmonofilament suture into internal carotid artery for90min and thenreperfused for24h. Paeoniflorin was injected intraperitoneally once-dailytreatment at30min or twice-daily treatment at30min and6h afterischemia. The rats in sham operation group and I/R group were injectedwith saline. After stability evaluation of rat MCAO modal by Lasterdoppler flowmetry, the neurological score was studied according to Longagrade5point standard. The infarct volume was detected by TTC stainingand brain water content was measured by dry and wet method. Meanwhile,pathological changes in hippocampal CA1region were observed throughHE staining. All the above methods were measured to determine itsneuroprotective effects, the optimal dosing concentration andadministration frequency.Results: Compared with the I/R group, neurological scores, theinfarction volumes and brain water content were significantly reduced and the pathological changes were observably improved after treatment withdifferent doses of paeoniflorin such as5mg kg-1、10mg kg-1、20mg kg-1, ofwhich20mg kg-1twice-daily treatment of paeoniflorin was mostsignificant.Conclusion: Treatment with paeoniflorin is beneficial to protect brainagainst ischemia/reperfusion injury. Morever, the experiment should be setup three different concentration of paeoniflorin such as5mg kg-1、10mgkg-1、20mg kg-1and paeoniflorin should be injected intraperitoneallytwice-daily treatment at30min and6h after ischemia.The second part: Effects of paeoniflorin on cerebral blood flow andcyclooxygense pathways against middle cerebral artery occlusioninjury in ratsMethods: A total of90♂SD rats were randomly divided into6groups:Sham operation group, cerebral ischemia-reperfusion model group（I/R gourp), low（5mg kg-1, twice）, middle （10mg kg-1, twice）and high（20mg kg-1, twice）doses of paeoniflorin groups and Nimodipine group.Relative regional cerebral blood flow (rCBF) was continuously monitoredover ischemic hemispheres by laser doppler flowmetry (LDF). Theexpression of COX-2in hippocampal CA1region was estimated byimmunohistochemistry and the contents of TNF-α, IL-1β, PGI2, TXA2andratio of PGI2/TXA2in frontal cortex of ischemic hemispheres weremeasured by ELISA kits. It’s the purpose that to investigate the neuroprotective effects and its mechanism of paeoniflorin on cerebral bloodflow and cyclooxygense pathways against Middle cerebral artery occlusioninjury in rats.Results: Paeoniflorin significantly increased rCBF relative to the I/Rgroup. In addition, paeoniflorin could inhibit COX-2expression and therelease of TNF-α, IL-1β and TXA2and prevent the down-regulation ofPGI2induced by I/R injury.Conclusion: The neuroprotective effects of paeoniflorin against focalcerebral ischemia-reperfusion rats might be attributed to passingblood-brain barrier (BBB) quickly, improving ischemic cerebrovascularflow velocity reserve and inhibiting arachidonic acid expression viacyclooxygense pathways.The third part: Neuroprotective effects of paeoniflorin on cerebralischemic rats by activating cannabinoid2receptorsMethods:105male SD rats were randomly divided into7groups:Sham operation group, cerebral ischemia-reperfusion model group, vehiclecontrol group, high（20mg kg-1,twice）doses of paeoniflorin groups,selective CB2receptor antagonist AM630group, paeoniflorin (20mg kg-1,twice) with AM630group, and selective CB2receptor agonist HU308group. The detection methods in this part are identical to the second part.It’s to investigate neuroprotective effects of paeoniflorin on cerebralischemic rats by regulating cannabinoid2receptors. Results: The protective effect of PF (20mg kg-1, twice) could be partlyabolished by pretreatment with AM630.Conclusion: The activation of cannabinoid2receptors might beinvolved in paeoniflorin induced neuroprotection in cerebral ischemia.