The Neuroprotective Effect And Mechanism Of Diminazene Aceturate Treatment Via Intraperitoneal Administration On Focal Cerebral Ischemic/Reperfusion Injury In Rats

Dysfunction after acute ischemic stroke poses a serious burden on the social economy.Timely opening of occluded blood vessels is an effective treatment.However,even after strict screening,injury of patients there is a lot of nerve function defect sustained only 12~34% patients but complex brain.With the gradual promotion of early reperfusion therapy,according to the law of the development of diseases targeted for reducing reperfusion therapy after cerebral ischemia / reperfusion injury method is to further reduce ischemic stroke related disability is an important task for neuro-researchers.Angiotensin II(Angiotensin II,Ang II),which is commonly found in organism,is an important material to mediate oxidative stress and promote inflammation.Cerebral ischemia / reperfusion injury was more severe in Ang mice,and blockade of angiotensin II type 1 receptor could reduce cerebral ischemia / reperfusion injury.These findings suggest that the angiotensin system may play an important role in the pathophysiology of cerebral infarction.In recent years,the discovery of angiotensin-converting enzyme 2(Angiotensin converting 2 enzyme,ACE2)can be transformed into Ang II and antagonist in function of angiotensin 1-7(Ang(1-7)),is considered to have protective effect on ischemic stroke.Aceturate(Diminazene,DIZE)is a potential ACE2 agonist.It has been found that intraperitoneal injection of DIZE can significantly reduce infarct volume and improve neurological function in rats after transient focal cerebral ischemia(transient focal ischemia),but the mechanism is not clear.The purpose of this study is to use a model more in line with the clinical acute large artery occlusion to evaluate the protective effect of DIZE on focal cerebral ischemic/reperfusion injury by intraperitoneal injection after reperfusiont and identify the best indications,and initiate an exploration of possible mechanisms.Part 1 Establishment and evaluation of rat model of focal cerebral ischemia / reperfusion injuryObjective: To establish a focal cerebral ischemia / reperfusion injury model in rats by intraluminal intraluminal occlusion and explore the relationship between duration of ischemia and reperfusion injury.Method:1.The relationship between ischemia time and reperfusion injuryThirty-two experimental rats were randomly divided into two groups,which were established the model of transient occlusion of middle cerebral artery for one hour or 2 hours,and the neurological function was observed at the same time after reperfusion for 24 hours.The following parameters were evaluated:(1)The mortality of rats in the model was successfully established within 24 hours(group n=16/);(2)The degree of neurological impairment score(NSS)and weight loss(n=16/ group);(3)TTC staining was used to evaluate the difference of infarct volume between the two groups(group n=10/);(4)the pathological features of brain tissue were observed by HE staining and Tunnel staining(group n=6/);2.The changes of MDA levels in brain tissue of rats with different ischemic time 24 rats were randomly divided into four groups,the same set of middle cerebral artery occlusion for 1 hours or 2 hours short model,two groups were in 6 hours or 24 hours after reperfusion were sacrificed and brain ischemia detection area MDA content;the other set up blank group(6 rats)as a baseline MDA.Result:1.The relationship between ischemia time and reperfusion injury(1)Sixty-eight rats were sacrificed in the study(2)NSS scores of 1 hour ischemia group were significantly lower than the 2 hour group(4.0[3.5,5.0] v.s.,10.5[8.5,11.5],p<0.001);and the weight loss was significantly lower than that of the latter(21.7 + 2.6 v.s.39 + 4.3,p<0.001)(g);(3)TTC showed that there was no significant difference between the two groups at the time of ischemia;(4)A large number of apoptotic cells were found in HE staining,and Tunel staining confirmed that apoptosis was the main mode of cell death in the ischemic area of the two groups.The percentage of Tunel positive cells in the ischemic group was higher than that in the ischemic group at 1 hours after ischemia2.The changes of MDA levels in brain tissue of rats with different ischemic time The level of MDA in cerebral cortex of ischemic 1 hours group was significantly higher than that in the 24 hours after ischemia,and the level of MDA in the ischemic group was lower than that of the 24 hour group at the same time of 6 hours after ischemia for 2 hours.Conclusions:1.Under the conditions of this study,the rat transient focal ischemia 1~2 hours after 24 hours of reperfusion,apoptosis is the main way of cell death in ischemic area;2.Under the conditions of this study,a transient ischemia there are significant differences between the 1 hours /2 hours in the model of oxidative stress injury the development speed of ischemia after 1 hours of reperfusion the level of lipid oxidation peak at sixth hours after reperfusion.Part 2 The neuroprotective effect and mechanism of Diminazene aceturate treatment via intraperitoneal administration on focal cerebral ischemic/reperfusion injuryObjective: To investigate the effects of intraperitoneal injection of DIZE on reperfusion injury in rats with two models of transient focal cerebral ischemia.Methods:1.The effects of intraperitoneal injection of DIZE on ischemia reperfusion injury following 1 hour MCAOAdult male SD rats were randomly divided into 5 groups: A(group n=10/),sham operation(simple neck incision,common carotid artery ligation and separation;B),ischemia 1 hours + intraperitoneal injection of normal saline;C,ischemia 1 hours + intraperitoneal injection of physiological DIZE 1.0mg/kg;D,ischemia 1 hours plus intraperitoneal injection.DIZE 5.0mg/kg E,physiological;ischemia 1 hours + intraperitoneal injection of physiological DIZE 15.0mg/kg.At the end of 24 hours after surgery,the NSS score was used to evaluate the neurological deficits in the five groups.The infarct size was assessed by TTC staining(%).In order to further protect the most significant effect of the lowest dose as the experimental dose,the sham operation group and normal saline as negative control and positive control;through the measurement of tissue around the biochemical reagent kit to detect infarct(MDA)concentrations reflect the level of oxidative stress(group n=6/);by Tunel staining detected in brain tissue of irreversible cell damage(the proportion of group n=6/).2.The effects of DIZE on ischemia reperfusion injury following 1 hour MCAOAdult male SD rats were randomly divided into 3 groups: A(group n=10/),sham operation(simple neck incision,common carotid artery ligation and separation;B),ischemia 1 hours + intraperitoneal injection of normal saline;C,ischemia 1 hours + intraperitoneal injection of physiological DIZE 5.0mg/kg.24 hours after the end of the study,the NSS score was used to evaluate the degree of neurologic impairment in the five groups.The infarct size was assessed by TTC staining.The level of lipid peroxidation was measured by detecting the level of MDA in ischemic brain tissue.3.The effect of DIZE via intraperitoneal administration on the activity of ACE2,Ang and Ang(1-7)The rats were divided into blank group,1 hours after ischemia and reperfusion for about sixth hours,and the rats were subjected to ischemia and reperfusion for 1 hours.The ACE2 activity,Ang II(1-7)and Ang(n=6)were detected by ELISA.Result:1,For one-hour-MCAO model,intraperitoneal injection of DIZE 5.0mg/kg group NSS score and infarct size was significantly lower than the normal saline control group,dose and protective effect of a U shape trend;intraperitoneal injection of DIZE 5.0mg/kg group MDA level was significantly lower than the saline control group;Tunel group received intraperitoneal injection of DIZE staining positive cells were significantly lower than normal the saline control group,and with caspase 3 level decreased;2.For the ischemia reperfusion model of,the NSS score,infarct size and MDA level of intraperitoneal injection of DIZE group were not significantly different from those of saline control group(5.0mg/kg);3,from the plasma concentration differences,group DIZE plasma Ang(1-7)were significantly higher than those in the saline group(1.260 + 0.449,0.861 + 0.401ng/ml,p<0.01),the other indexes had no significant difference between the two groups;while in the brain tissue of DIZE treatment group and saline control group showed no significant difference between the three indicators.Conclusion:1.Intraperitoneal injection of DIZE 5.0mg/kg at the time of reperfusion can reduce the degree of delayed lipid oxidative stress and apoptosis in rats;2.Elevation of Ang(1-7)level was observed in plasma but not in the ischemic cortex at 24 hour after DIZE administration.Part 3 The protective effect of DIZE on injury derived of glucose deprivation and reperfusionObjective: To establish a model of primary neuronal glucose deprivation / reperfusion(OGD/R)injury,and evaluate whether the protective effect is ACE2 dependant.Method:1.To culture primary cortical neurons of newborn rats and purify them;2.Evaluate he effect of DIZE on primary neuronal OGD/R damage.Neurons were divided into 5 groups,respectively: normal culture;OGD/R+ normal culture;the OGD/R +DIZE 0.1 M;the OGD/R +DIZE 1 M;OGD/R +DIZE,10 M,24 hours later by Annexin-PI double staining flow cytometry to evaluate the degree of apoptosis,Annexin-PI double negative and Annexin negative PI positive,two double positive cells as a proportion of grade data were compared between the two groups;by Western blot to detect the level of cl-caspase 3;detection of mitochondrial membrane potential TMRM fluorescent probe(MTMP);each index set of 3 parallel repetition.Result:1.Nissl staining showed that the neurons ratio is about 83.9 + 5.2%;2.Compared with the negative control group,cell apoptosis after OGD/R treatment significantly suppressed in DIZE groups.Western blot blank group cl-caspase 3 level was significantly higher than that of the normal culture group;3.DIZE significantly inhibited the decrease of MTMP,with a dose-dependent effect.Conclusion: DIZE can inhibit the apoptosis of primary neurons after glucose deprivation / reperfusion.