Effect of DL-3-n-butylphthalide on cognitive function and the regulating role of Nrf2/SIRT3 signaling pathway in vascular dementia mice

Objective To investigate the effects of butylphthalide(NBP) on cognitive function and Nrf2 / SIRT3 signal pathway in vascular dementia (VD) mice. Methods Wild-type mice (Nrf2+ /+ ) were divided into sham group, model group (VD group), butylphthalide treatment group (Nrf2+ /+ NBP group), and Nrf2 gene knockout mice (Nrf2-/-) were divided into Nrf2-/-model group (Nrf2-/-VD group) and Nrf2-/-treatment group (Nrf2-/-NBP group). Both the model group and the treatment group were repeated.The bilateral common carotid arteries were ligated three times to establish a mouse model of cognitive dysfunction caused by cerebral ischemia-reperfusion.The sham group only isolated the bilateral common carotid arteries and threaded the wires, but did not block blood flow.Morris water maze experiment was used to analyze the cognitive function of mice.HE staining was used to observe the changes of neuron morphology and structure in CA1 region of hippocampus, and immunohistochemical analysis was used to analyze the positive expression of caspase 3 and caspase 9 in mouse CA1 region of hippocampus.Western blot was used to detect mouse hippocampus Nrf2, p62, LC3, SIRT3 protein expression. Results (1) In Morris water maze experiment: compared with VD group, the escape latency of Sham group and Nrf2+ /+ NBP group was significantly shorter on the 5th day ((20.69±8.91) s, (7.58±9.47)s, (8.41±12.20)s; q=3.58, 5.07, both P 0.05). (2) Pathological results showed that, compared with VD group, the damage of pyramidal neurons in CA1 area of hippocampus in Sham group and Nrf2+ /+ NBP group was lighter, and that in Nrf2-/- VD group was more serious, and the improvement of neuron morphology was not obvious after NBP treatment.(3) The expression of apoptosis: compared with VD group, the expression of caspase-3 and caspase-9 in the CA1 area of hippocampus in Sham group and Nrf2+ /+ NBP group were significantly lower, and those in Nrf2-/-VD group were significantly higher (t=3.48, 2.95, 3.46, 2.93, -2.99, -3.77, all P 0.05). (4) Expression of related proteins: compared with VD group, Nrf2, SIRT3, p62 protein expression increased, LC3II/I ratio decreased in Nrf2+ /+ NBP group(t=-3.24, -4.04, -4.03, 3.62, all P<0.01); Nrf2, LC3II/ I ratio decreased, SIRT3, p62 protein expression increased in Sham group(t=3.44, 4.72, -3.52, -4.19, all P<0.01); Nrf2, SIRT3, p62 protein expression decreased and LC3II/I ratio increased in Nrf2-/-VD group(t=9.14, 4.20, 4.30, -3.78, all P<0.01); Compared with Nrf2-/- NBP, the expression of Nrf2, SIRT3, p62 decreased, and LC3II/I ratio increased in Nrf2-/-VD group(t=2.40, 3.24, 1.21, -1.16, all P<0.01). The expression of Nrf2, SIRT3, p62 protein in Nrf2+ /+ NBP group increased, and the ratio of LC3II/ I decreased (t=-3.29, -5.00, 6.24, all P<0.01). Conclusion Butylphthalide can reduce the apoptotic damage in hippocampus of VD mice and improve cognitive dysfunction caused by repeated ischemia-reperfusion injury.Regulating Nrf2 / SIRT3 pathway to inhibit hippocampal neuronal apoptosis and autophagy may be its role mechanism. Key words: Butylphthalide(NBP); Vascular dementia; Nrf2; SIRT3; Apoptosis autophagy