Nd-308 Anti-cerebral Ischemia - Reperfusion Injury And Its Mechanism Of Preliminary Discussion

Cerebrovascular disease is a clinical common,frequently encountered disease in the elderly.The rates of morbility,deformity and mortality are very high.It is one of the important diseases that lead significant harm to human health.Among them,the incidence of ischemic cerebrovascular disease is more accounted in the clinic.It is the focus and current research which is resume cerebral blood flow as soon as possible and,if possible, reverse the injury for nerve cells.But if we can not restore blood flow in time,it will lead brain ischemia and reperfusion to injury,and exacerbate the patient's conditions.Recently, it was found that cell injury is a dynamic,rapid development of the cascade after cerebral blood flow interruption and reperfusion of the brain.The injury cascade includes lots of pathological mechanisms of excitatory aminoacid toxicity,energy metabolism obstruction,intracellular calcium overload,inflammatory response and so on.At present, the main therapy for the ischemic stroke remain thrombolysis,anti-thromboxane and anticoagulation of platelet,which,although exert better effects on ischemic stroke in acute stage,sometimes produce untoward reactions such as hemolysis and so on, moreover demand firmly time window of therapy.As a result,there are urgent needs for novel therapy methods and drugs.ND-308,a novel derivatives of one nature drug synthesized by Shandong Engineering Research Center For the nature drug.was demonstrated in our former experiments having many pharmacologic actions such as extending coronary artery vessel,improving blood flow in ischemic area of heart, diminuting the scope of acute myocardial infarction,ameliorating the myocardial dysfunction induced by ischemia-reperfusion,and so on.In our present study,we investigate the effects of ND-308 on the injuries induced by cerebral ischemia-reperfusion and further approach the potential neuroprotection mechanisms of ND-308.PartⅠProtective effect of ND-308 on focal cerebral ischemia injury in ratsFocal cerebral ischemia-reperfusion(I/R) model was established by middle cerebral artery occlusion(MCAO) for 2 hours followed by 24h reperfusion.96 rats with MCAO were randomly divided into sham-operated group,model group,ND-308-treated (10mg/kg,20mg/kg) groups,and each group consisted of 24 rats.The rats of ND-308 treated groups were administrated ND-308(dissolved in 0.9%saline) by tail vein at 30 min and 6 h after reperfusion,and the rats of sham-operated group and model group were administrated the same dosage of 0.9%saline.After 24 h of reperfusion,the neurological deficit score of each rat was obtained according to Longa's method,by a single experimenter,who was blinded to the experimental treatment groups.Afterward,the rats were sacrificed,then the brains were isolated and the volume of infarction and the content of water in rats brain were measured.The same fraction of ischemic brain hemisphere were isolated and fixed in 4%paraformaldehyde,then paraffin sections were made and stained by hematoxylin-eosin(H&E) to observe the pathological change of ischemic brain tissue.Results showed that ND-308 could improve the neurological deficits(P＜0.05 or p＜0.01vs model group,n=8) and markedly decreased the area as well as the volume of the cerebral infarction in rats(P＜0.05 or p＜0.01,n=8).PartⅡThe mechanism of the protective effect of ND-308 on cerebral ischemia.1.Influence of ND-308 on cell apoptosis and related gene expression in rats20 rats with MCAO were randomly divided into sham-operated group,model group, ND-308-treated(10mg/kg,20mg/kg) groups,and each group consisted of 5 rats.In order to evaluate the effect of ND-308 on apoptosis of neurons in I/R rats, deoxynucleodyltransferase-mediated biotinylated UTP nickend labeling(TUNEL) assay was carried out to determine the apoptosis of neurons,furthermore the expression of apoptosis-related protein:bcl-2 and Bax were evaluated by immunohistochemistry. ND-308 could significantly attenuate the pathological lesion induced by I/R,inhibit apoptosis of neurons in ischemic penumbra(P＜0.05 or p＜0.01 vs model group,n=5) and could decrease the protein expression of Bax(P＜0.05 or p＜0.01 vs model group,n=5), while increase the expression of anti-apoptotic protein,Bcl-2(P＜0.05 or p＜0.01 vs model group,n=5).2.The anti-inflammatory effects on focal cerebral ischemia rats20 rats with MCAO were randomly divided into sham-operated group,model group, ND-308-treated(10mg/kg,20mg/kg) groups,and each group consisted of 5 rats.After 24 h of reperfusion,a serum sample was obtained,and then centrifuged for IL-1βlevel determinations,which were performed by enzymelinked immunosorbent assay(ELISA) as specified in manufacturer's kit.The same fraction of ischemic brain hemisphere were isolated for the measurement of the expression of TNF-α,NF-κB and IL-1βproteins by immuno-histochemical method.Compared with shame operation group,rats in I/R group exhibited obviously increased expression of IL-1βand TNF-α,the expression of NF-κB was also increased and translocated into the nuclear.ND-308 could remarkly down-regulate the expression of IL-1β,TNF-αand NF-κB,inhibite the translocation of NF-κB(p＜0.05 or 0.01 vs model group,n=5).Conclusion:ND-308 had obviously protective effects on focal cerebral ischemia-reperfusion injury in rats.Its neuroprotective mechanisms might be related to the effects of the extenuation of inflammatory reaction mediated by cytokines and inhibition of nerve cell apoptosis.