The Neroprotective Effects Of MD On Cerebral Ischemia Reperfusion Injury In Rats

The treatment of cerebral ischemia reperfusion injury (IRI) has been a worldwideproblem in the field of neuroscience, which remains unresolved at present. MD, aneffective ingredient extracted from a traditional Chinese medicine, was previously usedfor treating the myocardial ischemia-reperfusion injury with remarkable effectivenessand small side effects. In view of the partly common mechanism of injury between thecardial and cerebral IRI, therefore, a direct therapeutic effect on cerebral IRI would besuggested to be existed. Our present study was fousing on the evaluation of theneuroprotective effects on focal cerebral IRI and the exploration of the relatedpharmacological mechanisms, in order to provide theoretical guidance for therapy.Objective(1) To evaluate the effects of MD on cerebral IRI in rats;(2) To explore the mechanism of MD on the treatment of cerebral IRI.Method⑴Pharmacodynamics Evaluation①Established a classic focal cerebral ischemia-reperfusion injury model on rats byusing the suture method, namely, the middle cerebral artery occlusion (MCAO) ratmodel, and then implemented reperfusion after ischemia for2h；②Male SD rats were randomly divided into seven groups (n=12): The MD(30mg/kg) group, MD(60mg/kg) group, MD(120mg/kg) group, MK801group(2mg/kg), nimodipine and cyclophosphamide group (1mg/kg+50mg/kg), the modelgroup (normal saline1.5ml) and the sham group.These drugs were injected twice viathe tail vein at10min before reperfusion and1h after reperfusion.24h after reperfusion, the serum was kept and evaluated to cmpare the therapeutic effect ofMD on cerebral IRI by using major indices such as the neurological score,thepercentage of the brain infarction volume and pathology changes.⑵Pathology and the expression changes of the NR2A/2B mRNA and protein ofNMDA receptor subunit in cortex of MCAO rats at different time points afterischemia reperfusion injuryMale SD rats were randomly divided into6groups (n=8), including the shamcontrol group and the groups after IRI for6h、12h、24h、48h、96h respectively.Establish the MCAO model and take brain tissue at different time points afterreperfusion.2rats from each group were performed HE staining to observe thepathology changes, meanwhile, cortical tissue from the infarct zone of the other6rats was taken to detect the expression changes of the NR2A/2B mRNA and proteinof NMDA receptor subunit by using real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot.⑶Effects of MD on the SOD activity, the contents of MDA, GSH and NO in serum,and the protein expression of NR2A/2B,CREB and BDNF of MCAO rats at24h afterIRIMale SD rats were randomly divided into5groups (n=6), including MD (60mg/kg)group, MK-801group (2mg/kg), nimodipine and cyclopho-sphamide associatedtreatment group (1mg/kg+50mg/kg), IRI group and sham control group. All groupswere performed to establish the MCAO model except the sham control group,following by drug delivery with the same dosage and method as (2) above. At thetime point of24h after reperfusion, use the serum taken in step (1) to detect the SODactivity, the contents of MDA, GSH and NO in serum, and the protein expression ofNR2A/2B, CREB and BDNF in the infarction cortex area of each group. Result⑴Pharmacodynamics EvaluationCompared with IRI group, the neurological score and the percentage of cerebralinfarction of each treatment group were decreased notably, among which the reductionof the indices in the MD (60mg/kg) group was the most significant (P <0.01). MD couldsignificantly reduce the edema of neuronal cell and improve the functional status ofmitochondria and mitigate degranulation of endoplasmic reticulum and reduce theapoptotic cells.⑵Pathology and the expression changes of the NR2A/2B mRNA and protein ofNMDA receptor subunit in cortex of MCAO rats at different time points after ischemiareperfusion injuryThe pathology changes of MCAO rats were most severe in24h group comparing toother groups. The overall trend of NR2A/2B mRNA and protein expression wasdecreasing first and increasing afterwards, reaching the lowest point at the time point of24h after reperfusion (P<0.01). And the protein expression of NR2A/2B had nosignificant difference compared with the normal group at96h (P>0.05).⑶Effects of MD on the SOD activity, the contents of MDA, GSH and NO in serum,and the protein expression of NR2A/2B,CREB and BDNF of MCAO rats at24h afterIRI①MD could increase SOD activity and GSH content in serum（P<0.05/0.01）, as well asreducing the NO content (P<0.05).②At24h after reperfusion, the mRNA NR2A/2B expression raised significantly in MDgroup (P<0.05), and the NR2A protein expression increased as well.(P<0.05).③In addition, MD could up-regulate the mRNA expression of CREB and BDNF at24hafter reperfusion (P<0.01/0.05). ConclusionMD has shown clear protection and treatment effects on the cerebral IRI of MCAOrats. The possible mechanism could be listed as follows: it would restrain the oxidativestress by significantly enhancing the SOD activity and the GSH content in the serum; itwould confront the nitride stress by reducing the NO content in the serum; it wouldinhibit the excitotoxicity response resulting from the cerebral ischemia by up-regulatingthe NR2A mRNA and protein expression; and it would prevent neurons cells apoptosisby increasing the CREB and BDNF mRNA expression.