Edaravone Ameliorates Cognitive Impairment Through Improving Iron Dysmetabolic In Vascular Dementia Rats

Objective: Vascular dementia(VD)is a clinical syndrome of cognitive impairment which is caused by a series of cerebral vascular factors.Statistics have shown that VD is the second most common type of dementia after the Alzheimer's disease(AD).Chronic cerebral hypoperfusion(CCH)is one of the primary causes of VD,and the pathogenesis is not entirely clear and the effective treatment strateges are also lack.Recent study has found that iron metabolism disorder was observed in the process of CCH.Edaravone,as a free radical scavenger,can inhibit oxidative stress damages induced by CCH,but the function and mechanism of iron dysmetabolic have not to be clarified.In this experiment,we systematically investigated that the improvement of cognitive function and oxidative stress in the VD rats induced by CCH after edaravone treatment,and further explored the changes of iron content and iron transport factors.Methods: 3 months male Sprague-Dawley(SD)rats weighting in250-300 g were used as the research animals.The model of VD was be established by bilateral common carotid arteries occlusion(BCCAO).The rats were randomly divided into four groups: the sham group,vehicle group,edaravone3.0 group and edaravone6.0 group.The sham and vehicle group rats were provided 3.0 mg/kg saline twice a day,while 3.0 mg/kg or 6.0 mg/kg edaravone was used to the edaravone 3.0 and edaravone6.0 group rats after operation for 28 days.The behavioral tests were conducted after 28 days by morris water maze(MWM).The behavioral tests contain place navigation test and spatial probe test which were used to evaluate the learning and memory ability of rats.After the spatial probe test,morphological changes were examined by Nissl's staining in hippocampal CA1 of rats;reactive oxygen species(ROS)levelswere measured by biochemically,and iron content was determined by inductively coupled plasma mass spectrometry(ICP-MS)in the hippocampus;the levels of iron transport proteins including divalent metal transport 1(DMT1),transferrin receptor 1(Tf R1)and ferroportin 1(Fpn1)were respectively assessed by western blot and enzyme-linked immunosorbent assay(ELISA);Fpn1 m RNA and hepcidin m RNA levels were detected by reverse transcription-polymerase chain reaction(RT-PCR).Results:1 Behavior test results1.1 The place navigation test result: The escape latency was gradually shorten with the increased training days in all groups rats(F(4,144)=306.60,P<0.01).Starting from the second day of training,compared with the sham group rats,the escape latency was significantly prolonged in the vehicle group rats(P<0.01).However,the escape latency was remarkably attenuated after edaravone treatment as compared with the vehicle group(P<0.05).1.2 The spatial probe test result: Compared with the sham group,the ratio of time that the rats spent in the target quadrant was decreased in the VD rats(P<0.01).While the ratio of time that the rats spent in the target quadrant was significantly prolonged after the 3.0 mg/kg or 6.0 mg/kg of edaravone treatment as compared with the vehicle group(P<0.05,P<0.01).2 Nissl's staining resultsNissl's staining observed that neurons were arranged closely,and the neurones had normal morphology,integrated structure,clear nucleus in hippocampal CA1 of the sham group rats.However,in the vehicle group,markedly changes were discovered in above area,including neuronal cell loss,nuclei shrinkage,lightly stained neurons.But,these neuropathological signs were suppressed by treatment with edaravone.3 ROS levels resultsCompared with the sham group rats,the ROS levels were significantly increased in the hippocampus of the vehicle group rats(P<0.01).The ROS levels were obviously decreased after treating with edaravone as comparedwith the vehicle group rats(P<0.01).4 Iron contents resultsIron deposit was more abundant in the hippocampus of vehicle group rats as compared with the sham group rats(P<0.01).Although the iron content of the Edaravone3.0 group was lower than the vehicle group,no significant difference was found both of them(P>0.05).In addition,there was a significant difference in the iron content of hippocampus between the edaravone6.0 group and the vehicle group(P<0.01).However,no significant difference in iron content was found both the edaravone6.0 group and the edaravone3.0 group(P>0.05).5 Western blot results5.1 The expression of DMT1+IRE?DMT1-IRE and Tf R1 proteins in the hippocampus of VD ratsRats in the vehicle group expressed the DMT1+IRE protein level clearly higher than the sham group rats(P<0.05).But on the contrary,no significant difference in expression of DMT1-IRE protein was found between vehicle and vehicle groups(P>0.05).In addition,compared with the sham group,CCH also significantly increased the expression of Tf R1 protein(P<0.01).5.2 The effects of edaravone on the DMT1+IRE?DMT1-IRE and Tf R1 proteins expressionCompared with the vehicle group,the increased DMT1+IRE level was attenuated by the 3.0 mg/kg or 6.0 mg/kg of edaravone treatment(P<0.01,P<0.05,respectively).Meanwhile,edaravone 3.0 mg/kg or 6.0 mg/kg treatment remarkably also attenuated the up-regulation of Tf R1 protein in the BCCAO rats(P<0.05).But,no significant difference was found between the edaravone6.0 group and the edaravone3.0 group in terms of above proteins expression(P>0.05).6 ELISA resultsStatistical analysis showed that there was a significant difference about Fpn1 level in the hippocampus of four groups.Compared with the sham group rats,CCH considerably down-regulated Fpn1 level(P<0.05),which waseffectively restricted by 6.0 mg/kg of edaravone treatment(P<0.05).However,there was no significant difference for Fpn1 expression between the edaravone3.0 group and the edaravone6.0 group(P>0.05).7 RT-PCR resultsWe observed that hepcidin m RNA level was obvious increased in the vehicle group than the sham group(P<0.05).The 6.0 mg/kg body weight of edaravone treatment significantly inhibited the up-regulation of hepcidin m RNA in the hippocampus of rats after BCCAO(P<0.05).However,there was no significant difference about Fpn1 m RNA level among four groups(P>0.05).Conclusion:1 The BCCAO method is an ideal VD model of rats.2 Edaravone,as an antioxidant,can inhibit the oxidative stress and improve cognitive deficits in the hippocampus of VD rats.3 Edaravone can effectively improve the iron metabolism disorder in VD rats,and the mechanism may be related to the regulation of iron transport factors.