黄连素通过激活AMPK改善同型半胱氨酸诱导的大鼠痴呆和Tau蛋白过度磷酸化

Berberine ameliorates homocysteine-induced dementia and Tau hyperphosphorylation in rats by activating AMPK

目的:探讨黄连素(berberine,BBR)对同型半胱氨酸(homocysteine,Hcy)诱导的大鼠痴呆和Tau蛋白过度磷酸化的作用及机制。方法:36只SPF级雄性SD大鼠随机分为对照组、模型组和治疗组,每组12只。模型组和治疗组大鼠给予尾静脉注射Hcy(400μg·kg^(-1)·d^(-1))2周,对照组大鼠注射同剂量生理盐水(normal saline,NS);2周后治疗组大鼠用BBR(100 mg·kg^(-1)·d^(-1))灌胃10周,对照组和模型组大鼠用同体积的NS灌胃。Morris水迷宫实验检测大鼠的空间学习记忆能力;Western blot检测Tau蛋白的磷酸化(pT205和pS396)、腺苷酸活化蛋白激酶(AMPactivated protein kinase,AMPK)磷酸化(p-AMPK)及蛋白磷酸酶2A催化亚基(protein phosphatase 2A catalytic subunit,PP2Ac)磷酸化(p-PP2Ac)、去甲基化(DM-PP2Ac)水平和突触相关蛋白(PSD93和PSD95)的表达水平;免疫组化实验检测大鼠海马区pS396的表达水平;高尔基染色检测树突棘的密度。BBR或BBR联合Compound C(AMPK抑制剂)处理HEK293-Tau细胞(稳定转染人类全长Tau蛋白的人胚肾母细胞瘤细胞)24 h,CCK-8法检测细胞活力,Western blot检测pT205、pS396、AMPK、p-AMPK、PP2Ac、p-PP2Ac和DM-PP2Ac的表达水平。结果:(1)BBR显著改善Hcy大鼠的空间学习记忆能力(P<0.05);(2)BBR显著降低Hcy大鼠海马区Tau蛋白磷酸化水平并改善Hcy大鼠突触功能(P<0.05);(3)BBR显著增加Hcy大鼠海马区AMPK和PP2Ac的活性(P<0.05);(4)在HEK293-Tau细胞中,BBR显著降低Tau蛋白磷酸化水平(P<0.01);抑制AMPK活性可消除BBR对Tau蛋白的去磷酸化作用(P<0.05)。结论:黄连素通过激活AMPK降低Tau蛋白磷酸化水平并显著改善Hcy大鼠的认知功能。

AIM:To explore the effect of berberine(BBR)on homocysteine(Hcy)induced dementia and Tau hyperphosphorylation in rats and its mechanism.METHODS:36 SPF male SD rats were randomly divided into 3 groups(n=12):control group,model group and treatment group.The rats in the model group and the treatment group were injected with Hcy(400 mg·kg^(-1)·d^(-1))through the tail vein for 2 weeks,and the rats in the control group were injected with the same dose of normal saline(NS).After 2 weeks,rats in the treatment group were gavage with BBR(100 mg·kg^(-1)·d^(-1))for 10 weeks,and rats in the control group and model group were gavaged with the same dose of NS.Morris water maze test was used to detect the spatial learning and memory ability in rats.The expression levels of phosphorylated Tau(pT205 and pS396),AMP-activated protein kinase(AMPK)and its phosphorylation(p-AMPK),synapse associated proteins(PSD93 and PSD95),protein phosphatase 2 A catalytic subunit(PP2Ac)and its phosphorylation(p-PP2Ac)and demethylation(DM-PP2Ac)were tested by Western blot.The expression level of pS396 in rat hippocampus was detected by immunohistochemistry.The density of dendritic spines was detected by Golgi staining.In HEK293-Tau cells(human embryonic nephroblastoma cells stably transfected with human full-length Tau protein)were treated with BBR or BBR combined with Compound C(AMPK inhibitor)for 24 h.The viability was detected by CCK-8 method,and Western blot was used to test the levels of pT205,pS396,AMPK,p-AMPK,PP2Ac,p-PP2Ac and DM-PP2Ac.RESULTS:(1)BBR significantly improved the spatial learning and memory ability of Hcy rats(P<0.05);(2)BBR significantly reduced the level of phosphorylated Tau in the hippocampus of Hcy rats and improved synaptic function in Hcy rats(P<0.05);(3)BBR significantly increased the activities of AMPK and PP2Ac in the hippocampus of Hcy rats(P<0.05);(4)In HEK293-Tau cells,BBR significantly reduced the level of phosphorylated Tau(P<0.01).Inhibition of AMPK activity eliminated the dephosphorylation of Tau by BBR(P<0.05).CONCLUSION:BBR significantly reduced the level of phosphorylated Tau and improved cognitive function in Hcy rats through activating AMPK.