| Objective:Vascular dementia（VD）is a kind of acquired intelligence impairment syndrome caused by brain blood flow（CBF）reduction or blockade caused by cerebrovascular diseases,which mainly results in learning and memory dysfunction.Studies have shown that butylphthalide can improve cognitive dysfunction,but its specific mechanism has not yet been fully elucidated.In this experiment,the role of butylphthalide on the nuclear transcription factor 2（Nrf2）/silencing regulator 3（SIRT3）pathway was observed by establishing in a VD mouse model.Methods:In this study,male mice with ICR background were used to establish a mouse model of cognitive impairment induced by cerebral ischemia-reperfusion by ligating bilateral common carotid arteries three times.Butylphthalide was given intragastrically once a day for 28 days.The mice were divided into six groups:Nrf2+/+sham operation group（sham group）,Nrf2+/+model group(Nrf2+/+VD group),Nrf2+/+butylphthalide low dose treatment group(Nrf2+/+NBP-L group),Nrf2+/+butylphthalide High-dose treatment group(Nrf2+/+NBP-H group)and Nrf2-/-model group(Nrf2-/-VD group)and Nrf2-/-butylphthalide treatment group(Nrf2-/-NBP group)on Nrf2knockout mice.The Morris water maze test was used to analyze the cognitive function of each group of mice,including navigation experiments and space exploration experiments.The morphological changes of neurons in the hippocampal CA1 region were observed by HE staining.The positive expression of caspase3 and caspase9 in hippocampal CA1 region was analyzed by immunohistochemistry.The expression of Nrf2,SIRT3,P62 and LC3 in hippocampus of mice was detected by Western blot.Results:1 The results of Morris water maze1.1 NBP can improve cognitive function in VD miceCompared with the sham group,the escape latency（time of entering the water to find the platform）of Nrf2+/+VD group was significantly prolonged,and the percentage of staying time in the target quadrant（the quadrant of the original platform）was significantly reduced,and the difference was statistically significant（P<0.05）.Compared with Nrf2+/+VD group,the escape latency of Nrf2+/+NBP-L group and Nrf2+/+NBP-H group was significantly shortened,and the percentage of target quadrant residence time increased significantly,the difference was statistically significant（P<0.05）.Compared with the Nrf2+/+NBP-L group,the Nrf2+/+NBP-H group had shorter escape latency and longer target quadrant retention time,but the difference was not statistically significant（P>0.05）.1.2 Effects of NBP on Cognitive Function of Nrf2+/+and Nrf2-/-VD miceCompared with the Nrf2+/+VD group,the escape latency of the Nrf2-/-VD group was prolonged;the percentage of target quadrant residence time decreased,and the difference was statistically significant（P<0.05）.Compared with the Nrf2-/-VD group,the escape latency of the Nrf2-/-treatment group was shortened,and the percentage of target quadrant stay time increased,but the difference was not statistically significant（P>0.05）.In the Nrf2+/+treatment group,the escape latency was significantly shortened compared with the Nrf2-/-treatment group,and the percentage of target quadrant stay time was significantly increased,and the difference was statistically significant（P<0.05）.2 The observation of HE stainingCompared with the Nrf2+/+sham group,the vertebral neurons in the hippocampal CA1 area of the Nrf2+/+VD group were loosely arranged,the layer was poor,and the nucleus was pyknotic.After NBP treatment,the injuries of vertebral neurons in CA1 area were reduced,and the structure of neurons was more complete.However,there was no significant difference between NBP-H group and NBP-L group.Compared with Nrf2+/+VD mice,the morphological structure damage of vertebral neurons in hippocampal CA1area of Nrf2-/-VD mice was more serious,scattered,and the distribution of intact neurons was less,and a large number of nuclear pyknosis appeared.The improvement of neuronal morphology in Nrf2-/-VD mice after NBP treatment was not obvious.3 The results of ImmunohistochemistryCaspase3 and Caspase9 positive cells were expressed in the cytoplasm and showed a brownish yellow color.Compared with sham group,the expression of CA1 positive cells in the hippocampus of Nrf2+/+VD group was significantly increased（P<0.01）,but the expression of Caspase 3 and Caspase9 positive cells was significantly decreased after the treatment of NBP（P<0.01）.However,there was no significant difference in the expression of NBP-H group compared with NBP-L group（P>0.05）.Compared with Nrf2+/+VD group,Caspase3 and Caspase9 positive cells in Nrf2-/-VD group expressed more（P<0.01）,and there was no significant difference in Caspase 3and Caspase9 positive after the treatment of NBP（P>0.05）.4 Western blot results4.1 Impact of NBP on the Nrf2/SIRT3 pathwayCompared with sham group,the expression of Nrf2 protein in Nrf2+/+VD group was significantly increased（P<0.01）,and the expression of SIRT3protein was significantly decreased（P<0.01）.After NBP treatment,the expression of Nrf2 and SIRT3 in hippocampus of VD mice increased（P<0.01）.Compared with Nrf2+/+VD mice,Nrf2、SIRT3 protein in Nrf2-/-VD mice was significantly decreased in Nrf2 knockout mice（P<0.01）,and the proteins of Nrf2 and SIRT3 were significantly elevated in hippocampus after NBP treatment（P<0.01）,but the expression of Nrf2 and SIRT3 in Nrf2-/-VD+NBP treatment group was significantly lower than that in Nrf2+/+VD+NBP treatment group（P<0.01）.4.2 Changes in autophagy-associated proteins P62 and LC3Compared with sham group,the expression of P62 protein in Nrf2+/+VD group was significantly decreased（P<0.01）,and the ratio of LC3II/I was significantly increased（P<0.01）.After the treatment of NBP,the expression of P62 protein in hippocampus of VD mice was significantly increased（P<0.01）,the ratio of LC3II/I decreased significantly（P<0.01）.Compared with Nrf2+/+VD mice,P62 protein in Nrf2-/-VD mice was significantly reduced（P<0.01）and the ratio of LC3II/I was significantly increased（P<0.01）in Nrf2knockout mice,but there was no significant difference in the expression of P62 protein and the ratio of LC3II/I in hippocampus after the treatment of NBP.Conclusions:Butylphthalide can alleviate the apoptotic damage in the hippocampus of VD mice and improve cognitive dysfunction caused by repeated ischemia-reperfusion injury.Over-activation of apoptosis and autophagy plays an important role in the pathogenesis of VD.Butylphthalide can improve the damage of spatial learning and memory and pathological damage in hippocampus of VD mice.It may be one of the mechanisms of action to inhibit neuronal apoptosis and autophagy in hippocampus by regulating Nrf2/SIRT3 pathway. |
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