| Objective: Vascular dementia(VD)is the late stage of vascular cognitive impairment(VCI).With the increasing of aging population and improvement of living standards,the proportion of elderly people is increasing.As a common disease of elderly people,VD has brought a heavy burden to the families and society.In order to provide appropriate treatments for clinical medicine,we should have a better understanding of the pathogenesis of VD.It has been suggested that oxidative stress induced by chronic cerebral hypoperfusion(CCH)plays an important role in the pathogenesis of VCI.While nuclear factor erythroid 2-related factor 2(Nrf2)-antioxidant response element(ARE)signaling pathway is one of the most important anti-oxidative stress pathways.We established a widely accepted VD model of rats by permanent bilateral common carotid artery occlusion(BCCAO).In our study,it is explored that whether dl-3n-butylphthalide(NBP)could improve cognitive function in rats with VD,and discussed whether its protective mechanism is related to activating Akt/Nrf2 signaling pathway and regulating NOX1 in the hippocampus of rats.Methods:3-month-old male Sprague–Dawley rats weighing 250-300 g were randomly divided into four groups: Sham group,vehicle group,NBP low-dose group(NBP-L),NBP high-dose group(NBP-H).Permanent bilateral common carotid artery occlusion was used to establish VD model of rats.From the next day of the operation,sham group and vehicle group were provided with an equal volume of corn oil,while NBP-L and NBP-H were treated with 40mg/kg and 80mg/kg respectively through oral treatment.The treatments will last 28 days.After 4 weeks,the Morris water maze test was implemented at day 29-34,including the place navigation test and the spatial probe test.Respectively,they were used to evaluate the abilities of spatial learning and memory of rats.Then,HE staining was selected to observe the the pathological changes in the hippocampus of rats.To detect the expressions of t-akt,p-akt,t-Nrf2,n-Nrf2,HO-1,NOX1,Bcl-2,Bax,western blot was used.Additionally,immunohistochemical method was applied to observe the change of HO-1.Results:1 The behavioral test of Morris water maze1.1 The place navigation test: the escape latency of all the rats was gradually shortened with training.Compared with the sham group,the escape latency of rats in VD vehicle group was significantly prolonged(P<0.01).However,the poor performance was alleviated after the treatment of NBP.The two treatment group by NBP,showed shorter escape latencies in comparison with vehicle group,with statistic significance(P<0.01).1.2 The spatial probe test: There was significant difference in the ratio of time that rats spent in the target quadrant among four groups.Compared with the sham group,the vehicle group spent less time in the target quadrant,with statistic significance(P<0.01).While the deficits were significantly improved with treatment of NBP(P<0.01).2 The observation of HE stainingThe pyramidal cells in the hippocampal CA1 areas of the sham group arranged orderly and densely.There was no obviously loss in the number of neurons.The size of cells was normal and the morphology was integrity.The nucleolus and cytoplasm were clear.Compared with the sham group,there was significantly loss in neurons in vehicle group.The arrangement of pyramidal neurons was loosely and irregularly.Some neurons turned karyopyknosis,and cytoplasm hyperchromatic.After the intervention of NBP,the morphological changes described above had improved.The loss of neurons in CA1 area of hippocampus reduced,but there still exits a little amount of karyopyknosis.3 Western blot results:3.1 The effects of NBP on the Akt/Nrf2/HO-1 signaling pathwayThere was no statistical significance about the expression of t-akt among four groups(P>0.05).Compared with the sham group,the expression of p-akt,t-Nrf2,n-Nrf2,HO-1 in vehicle group decreased significantly(P<0.01,P<0.01,P<0.01,P<0.01)).While compared with the vehicle group,the expression of p-akt in NBP-L and NBP-H had increased obviously(P <0.05,P <0.01).And there was a significantly increase in the level of t-Nrf2、n-Nrf2 of NBP-treatment group(P<0.01,P<0.01).Similar,the expression of HO-1 in NBP-L and NBP-H also increased with statistical significance(P<0.05,P<0.01).But there was no statistical significance between NBP-L and NBP-H.3.2 The effect of NBP on the expression of NOX1 in hippocampus of ratsCompared with the sham group,the expression of NOX1 in vehicle group decreased significantly(P<0.01).However,after intervened with NBP,the decrease was obviously alleviated(P<0.01).Compared with the vehicle group,there was a significantly increase in the expression of NOX1 in NBP-treatment group(P<0.01).And the increase in NBP-H was more significantly(P<0.05).3.3 The effect of NBP on the expression of Bcl-2、Bax in hippocampus of ratsCompared with sham group,the expression of Bcl-2 decreased(P<0.01)while the expression of Bax increased(P<0.01)significantly in vehicle group,thus the ratio of Bcl-2/Bax was markedly reduced(P<0.01).After administration with NBP,the downregulation of Bcl-2 and upregulation of Bax reversed significantly(P<0.01,P<0.01).Consequently,the ratio of Bcl-2/Bax also increased with statistical significance(P <0.01).4 Immunohistochemistry results:Compared with sham group,the immunoreactive expression of HO-1 in CA1 neurons of vehicle group was significantly decreased(P<0.01).The positive expression product was brown particles.In comparision with vehicle group,with the treatment of NBP,there was an obviously increase in the positive expression of HO-1 in hippocampus of rats(P<0.05).Conclusions: Oxidative stress induced by chronic cerebral hypoperfusion may be an important factor contributing to VCI.Administration of NBP in the early time of chronic cerebral hypoperfusion could not only improve cognitive impairments in rats with VD,but also inhibit the apoptosis of neurons.It is supposed that its neuroprotection may be related to activate Akt/Nrf2 signaling pathway and regulate the expression of NOX1 in the hippocampus of rats. |
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