| Cerebral ischemic injury,as a pathophysiological process,having a serious damage to the human health,seriously affected people's survival time and the quality of life.The edaravone to serve as a kind of commonly used clinical brain protective agent,can remove the free radicals,inhibit the lipid peroxidation and delay neuronal cells death of brain,so as to reduce the degree of cerebral ischemia and nerve protective effect into full play.In recent years,autophagy has becoming a research hotspot of the cerebral ischemic diseases and got widespread attention.Autophagy is a kind of the catalytic process of degradation by themselves the damaged proteins and organelles mediated by the lysosomes.But the research about the edaravone influence on autophagy in brain tissues at present is not yet clear.The experiment is to through the use of the edaravone,research the influence of edaravone on the expression of autophagy in cerebral ischemia rats and to explore the edaravone plays a role of brain protection whether through regulating the autophagy progess.Objective:The ischemic models were established by the middle cerebral artery occlusion(MCAO)using suture-occluded method,and given drug intervention after confirming the success of the focal cerebral ischemia models.The tissue samples of brain were stained with HE to observe the pathological changes;and 2,3,5-triphenyltetrazolium chloride(TTC)straining was used to observe the cerebral infarcts and to detect the cerebral infarction volumes.The Atg-5protein and Beclin1 protein in the cerebral tissues of rats after ischemia were detected by Western-blot,and the expression level of the Atg-5 m RNA and Beclin1 m RNA were explored by RT-PCR.The aim is to research the effect of edaravone(eda)to autophagy of rats in brain with focal cerebral ischemia.Methods:1.72 healthy male and clean SD rats,weighing 250-330 g,were randomly divided into the control group,the experimental group and the eda group,with24 rats in each group.The MCAO model is established by reference to the Zea-Longa method.The rats in the experimental group were performed MCAO surgery,while the rats in the eda group were given eda intervention after the MCAO at the same time;the rats in the control group experienced the same operation in addtion to insert the line.The judging standard of the rats model's success was to reference to Zea-Longa.Each group was divided into three time points,namely 1 day,3 days and 7 days according to the different ischemic time with 8 rats subcutaneously after the MCAO surgery.To add the rats ramdonly in the process of MCAO,and ensure that the rats number in each group remains the same.2.Using HE staining to observe the pathological changes of brain tissues in each group.3.By using TTC method to observe the cerebral infarcts and to detect the cerebral infarction volumes.4.Detecting the expression level of Atg-5 protein and Beclin1 protein in the cerebral tissues by Western method.5.The expression level of Atg-5 m RNA and Beclin1 m RNA were evaluated by RT-PCR in rats.Results:1.The results of the neurological defect score:The scores of the control group at 1d,3d and 7d were all 0;the scores of each time point in experimental group respectively were(2.80±0.42)point,(2.87±0.35)point and(2.75±0.32)point;the scores of three time points in the edaravone group were:(2.03±0.47)point,(1.84±0.41)point and(1.97±0.37)point.Compared with the experimental group,the scores in edaravone group were differently decreased(P<0.05).2.The pathological morphological changes in the cerebral tissue in each group was observed by HE staining:The nerve cells in the control group were arranged in rules,clear boundary,the nucleus were complete;the neuron tissues in the expermental group had a patchy necrosis and clear boundary with the surrounding normal tissues,the structure was loose,and the nerve cells arranged in a mess,cell lineage was disordered,boundary was not clear,part of the nucleus dissolved,the above changes at 7d were the most obvious.The above changes in the eda group were reduced apparently when compared with that in the experimental group.3.The results of TTC method to observe the cerebral infarcts and to detect the cerebral infarction volumes:(1)The control group had no cerebral infarction lesions,while the other two groups had visible cerebral infarcts,and at the same time the infarction lesions were significantly reduced in the eda group than the experimental group;(2)The cerebral infarction volumes in three groups successively were 0mm3,(167.80±11.03)mm3,(119.40±12.12)mm3.The infarct volumes both in the experimental group and the eda group were remarkbly increased than that in the control group(P<0.05);compared with the experimental group,the cerebral infarction volume in the eda group had a significant decrease(P<0.05).4.The expression levels of the Atg-5 protein and Beclin1 protein in neuron tissues in all groups were:(1)The expression level of Atg-5 at 1d in the control group,the experimental group and the eda group respectively was:0.46±0.02,0.97±0.05,0.73±0.03.The level at 3d in each group successively was 0.39±0.08,1.57±0.06,0.97±0.05.The expression level at 7d in each group in sequence was 0.41±0.04,0.72±0.08,0.58±0.07.(2)The level of Beclin1 at 1d in three groups respectively was 0.72±0.02,1.64±0.18,0.91±0.09.The level at3 d in sequence was 0.67±0.04,2.18±0.17,1.64±0.18.The expression level at 7d was 0.69±0.05,1.02±0.24,0.82±0.17.The level of Atg-5 protein and Beclin1 protein in the control group at three time points had no statistical differences(P>0.05).The Atg-5 protein and Beclin1 protein in experimental group had the same expression tendency with the eda group,namely the level increased at 1d,arrived at the peak at 3d,reduced at 7d,but still had a higher level than the control group(P<0.05).Compared with the control group,the protein of Atg-5 and Beclin1 at each time point in experimental and eda group was significantly increased(P<0.05).The levels of Atg-5 protein and Beclin1 protein at each time point in eda group were remarkbly reduced compared with that in the experimental group(P<0.05).5.The Atg-5 and Beclin1 m RNA expressions were observed by RT-PCR in each group in every group:(1)The level of Atg-5 m RNA at 1d in the control group,the experimental group and the eda group respectively was:0.46±0.04,1.51±0.05,1.01±0.04,the level at 3d in each group was 0.55±0.03,1.99±0.09,1.41±0.06,the level at 7d in each group was 0.51±0.02,1.20±0.04,0.89±0.06.(2)The level of Beclin1 m RNA at 1d in all groups respectively was:0.76±0.04,1.68±0.12,1.02±0.07,the level at 3d was 0.71±0.05,2.05±0.12,1.51±0.10,the level at 7d was 0.85±0.06,1.41±0.20,1.02±0.11.The level of Atg-5 and Beclin1 m RNA in the control group at each time point had no statistical differences(P>0.05).The Atg-5 m RNA and Beclin1 m RNA in the experimental group had a similar trend with the eda group,namely the level increased at 1d,arrived at the peak at 3d,reduced at 7d,but still had a higher level than that in the control group(P<0.05).Compared with the control group,the m RNA of Atg-5and Beclin1 at each time point in experimentalgroup and eda group was significantly increased(P<0.05).The levels of Atg-5 and Beclin1 m RNA at each time point in eda group were remarkbly reduced compared with that in the experimental group(P<0.05).Conclusions:1.There were autophagy in normal neuron of rats,low experssion of Atg-5and Beclin1 were detected.2.Autophagy was activated after focal cerebral ischemic injury in rats.Its activation was manifested by the elevation of Atg-5 and Beclin1 both the protein and m RNA.3.Edaravone could inhibit the excessive activation of autophagy and relieve the nerve function damage in cerebral ischemic rats,protecting effect to the occurrence of cerebral ischemic injury.It may be one of the protection mechanisms of Edaravone. |
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