Effects Of Amifostine On Oxidative Stress,Mitochondrial Injury And Neuroprotection In Cerebral Ischemia-Reperfusion Mouse Model

Objective:The neuroprotective effect of amifostine was preliminarily observed through the changes of neurological deficit score(mNSS score)and cerebral infarct area after cerebral ischemia-reperfusion injury in mice.The neuroprotective mechanism of amifostine was explored from the aspects of oxidative stress/mitochondrial injury and the expression of apoptosis-related proteins.Methods:A total of 84 10-12-week-old C57BL/6 male mice aged 10-12 weeks were randomly divided into sham operation group(n=12),MCAO/R(Middle cerebral artery occlusion/reperfusion)group(n=24),MCAO/R group+low-dose-amifostine group(n=24),MCAO/R group+high-dose-amifostine group(n=24).The modified Zea-Longa method was used to embolize the right middle cerebral artery.The treatment group was administered once 30 minutes before embolization(the low and high dose were 200 mg/kg and 400 mg/kg respectively),and reperfused after 1 h of embolization.Brain tissues of each group were collected for experiments after 24 h of reperfusion.To explore the protective effect of amifostine on brain tissue by modified Neurological Severity Scores(mNSS),TTC staining and Nissl staining,and to explore the effect of amifostine on oxidative stress/mitochondrial injury by detecting the changes of oxidative stress indexes(Superoxide dismutase,SOD、Malonaldehyde,MDA)in ischemic hippocampal tissue and the expression of cytochrome-c(Cyt-c)in cytoplasm and mitochondria of hippocampal tissue.The protection of apoptotic neurons was observed by TUNEL/NeuN fluorescence double staining,and the expression of apoptosis-related proteins(Bax,Bcl-2,cleavedcaspase9,cleavedcaspase-3,p38MAPK,p-p38MAPK)was detected by Western blotting(Western Blot)to explore the potential mechanism of neuroprotective effect of amifostine.Results:1、Neurological impairment score:The mNSS score in MCAO/R group was significantly higher than that in Sham group(p<0.05),the mNSS score in L-amifostine group was lower than that in MCAO/R group,but there was no significant difference(p>0.05),and the score in H-amifostine group was significantly lower than that in L-amifostine group(p<0.05).2、Cerebral infarct area:The infarct size in L-amifostine group and H-amifostine group was less than that in model group(p<0.05),and the infarct size in H-amifostine group was significantly lower than that in L-amifostine group(p<0.05).3、Nissl staining:The Nissl bodies of neurons in hippocampal CA1 region of Sham group showed obvious blue-purple,complete cell structure,clear nucleus and obvious staining;neurons in hippocampal CA1 region of MCAO/R group shrank,the number of Nissl bodies was significantly less than that of Sham group,the structure was blurred,and the staining was slightly lighter;the number of Nissl bodies increased in L-amifostine group,and it increased more significantly in H-amifostine group,and the arrangement of Nissl bodies was compact and stained obviously.4、Oxidative stress:Compared with the Sham group,the SOD level in the hippocampus of the MCAO/R group was significantly reduced(p<0.05),and the MDA level was significantly increased(p<0.05);compared with the MCAO/R group,the SOD content in L-amifostine group and H-amifostine group increased(p<0.05),and the MDA content decreased(p<0.05);the content of SOD in H-amifostine group was higher than that in L-amifostine group,while the content of MDA in H-amifostine group was lower than that in L-amifostine group(p<0.05).5、Western blot results:Compared with the Sham group,the expression of mitochondrial cytochrome-c in the hippocampus of the MCAO/R group was reduced(p<0.05),and the expression of cytoplasmic cytochrome-c was increased(p<0.05),the expression of Bax,cleaved caspase-3,cleaved caspase-9 and p-p38 increased(p<0.05),and the expression of Bcl-2 decreased(p<0.05);compared with the MCAO/R group,the expression of cytochrome-c in the mitochondria of the L-amifostine group and H-amifostine groups increased(p<0.05),the expression of cytochrome-c in the cytoplasm decreased(p<0.05),the expression of Bax,cleaved caspase-3,cleaved caspase-9,and p-p38 decreased(p<0.05),and the expression of Bcl-2 increased(p<0.05);the expression of each index in L-amifostine group and H-amifostine groups were statistically different(p<0.05);the expression of p38 in each group had no statistical difference(p>0.05).6、TUNEL/NeuN fluorescent double staining:Compared with Sham group,the number of apoptotic neurons in hippocampal CA1 region of MCAO/R group increased and the number of surviving neurons decreased significantly(p<0.05);Compared with MCAO/R group,the number of apoptotic neurons in L-amifostine group was decreased,but the difference was not statistically significant(p>0.05),and the number of surviving neurons increased(p<0.05);the number of apoptotic neurons decreased significantly and the number of surviving neurons increased significantly in H-amifostine group,and the difference was statistically significant(p<0.05).Conclusion:1、Pretreatment with amifostine can significantly reduce the area of cerebral infarction,improve the neurological deficit and play a protective role in cerebral ischemia-reperfusion mouse model.2、Amifostine can up-regulate the expression of SOD,improve mitochondrial function and reduce the release of Cyt-c,mediate p38MAPK pathway,regulate the expression of apoptosis-related proteins,so as to exert anti-oxidant brain protective effect in cerebral ischemia-reperfusion mouse.