二苯乙烯苷通过调节GSK-3β/PP2A活性干预Aβ_(25-35)致拟痴呆大鼠模型Tau蛋白磷酸化进程

The intervention of TSG in Tau protein phosphorylation of the Aβ_(25-35)-induced dementia-like rat model by regulating GSK-3β/PP2A activity

目的探讨二苯乙烯苷(2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glycoside,TSG)通过调节Aβ_(25-35)致拟痴呆大鼠模型脑组织中糖原合成酶激酶3β(GSK-3β)、蛋白磷酸酯酶-2A(PP2A)、环磷酸腺苷依赖的蛋白激酶(PKA)的表达干预Tau蛋白磷酸化的机制。方法采用Morris水迷宫定位航行实验对50只24月龄老年雄性SD大鼠进行筛选,剔除天生痴呆大鼠后,筛选出36只,随机分为正常组、假手术组、模型组、TSG低剂量组(0.033g/kg)、TSG中剂量组(0.1g/kg)、TSG高剂量组(0.3g/kg),每组6只。大鼠手术造模14d后,TSG各剂量组大鼠按相应剂量灌胃药物(连续28d);其余3组大鼠每天灌胃等量生理盐水。灌胃结束后,采用避暗箱实验对大鼠学习记忆能力进行检测;免疫荧光法(IF)检测各组大鼠脑组织GSK-3β蛋白的表达;实时荧光定量聚合酶链式反应法(qRT-PCR)检测GSK-3β、PP2A、PKA mRNA表达情况;蛋白免疫印迹法(Western blot)检测Tau、PP2A、PKA蛋白表达情况。结果正常组、假手术组及TSG各剂量组大鼠避暗箱实验的潜伏期及错误次数均无明显变化;脑组织中GSK-3β、PP2A、PKA mRNA表达及GSK-3β、Tau、PP2A、PKA蛋白表达均无明显变化;模型组大鼠避暗箱实验的潜伏期明显下降(P<0.01),错误次数明显增多(P<0.01);模型组大鼠脑组织中GSK-3β、PKA mRNA表达水平明显升高(P<0.05),PP2A mRNA表达水平明显下降(P<0.05);GSK-3β、PKA及Tau蛋白表达水平明显升高(P<0.05或P<0.01),PP2A蛋白表达水平明显下降(P<0.01)。与模型组比较,TSG各剂量组大鼠避暗箱实验的潜伏期均出现明显升高(P<0.05或P<0.01),错误次数均明显减少(P<0.01);TSG各剂量组大鼠脑组织中GSK-3β、PKA mRNA表达水平均出现明显下降(P<0.05),PP2A mRNA表达水平均明显升高(P<0.05);TSG中剂量组大鼠脑组织中皮层区GSK-3β蛋白表达水平明显下降(P<0.05),TSG中剂量组及TSG高剂量组大鼠脑组织中海马区GSK-3β蛋白表达水平明显下降(P<0.01);TSG中剂量组及TSG高剂量组大鼠脑组织中PKA蛋白的表达水平明显降低(P<0.05);TSG各剂量组大鼠脑组织中Tau蛋白的表达水平均明显下降(P<0.05或P<0.01),PP2A蛋白的表达水平均明显升高(P<0.05或P<0.01)。结论TSG可以通过调节GSK-3β/PP2A活性干预Aβ_(25-35)致拟痴呆大鼠模型Tau蛋白磷酸化进程发挥抗痴呆作用,其机制可能与下调GSK-3β及PKA的表达和上调PP2A的表达有关。

Objective To explore the intervention mechanism of 2,3,5,4’-tetrahydroxystilbene-2-O-β-Dglycoside(TSG)in Tau protein phosphorylation by regulating the expressions of glycogen synthase kinase 3β(GSK-3β),protein phosphatase-2A(PP2A)and cyclic adenosine phosphate dependent protein kinase(PKA)in the brain tissues of the Aβ_(25-35)-induced dementia-like rat model.Methods Navigation experiment in the Morris water maze was performed to screen 5024-month-old male SD rats.After the rats with congenital dementia were removed,36rats were screened out.They were randomly divided into the normal group,the sham operation group,the model group,the low-dose TSG(0.033g/kg),medium-dose TSG(0.1g/kg),and high-dose TSG(0.3g/kg)groups,with 6rats in each group.After being surgically modeled for 14days,rats in each TSG group were given corresponding doses of drugs by intragastric administration(for 28consecutive days).The other three groups were given intragastrically the same amount of normal saline every day.After gavage,the learning and memory ability of the rats was tested by dark box experiment;the protein expression of GSK-3βby immunofluorescence assay(IF).The mRNA expressions of GSK-3β,PP2Aand PKA were detected by real-time fluorescence quantitative polymerase chain reaction(qRT-PCR).Western blot was adopted to detect the protein expressions of Tau,PP2Aand PKA.Results There was no significant change in the incubation period and number of errors showed by the dark box experiment in the normal group,the sham operation group and each TSG group.And their mRNA expressions of GSK-3β,PP2A,PKA and protein expressions of GSK-3β,Tau,PP2A,PKA in brain tissues did not significantly change.The model group had significantly decreased incubation period and significantly increased number of errors in the dark box experiment(P<0.01).They also had significantly increased mRNA expressions of GSK-3βand PKA(P<0.05)and significantly decreased mRNA expression of PP2A(P<0.05).Their protein expressions of GSK-3β,PKA and Tau significantly increased(P<0.05or P<0.01),while the protein expression of PP2Adecreased significantly(P<0.01).Compared with the model group,all the TSG groups had significant increase in the incubation period(P<0.05or P<0.01),but significant decrease in number of errors(P<0.01)as well as significant decrease in mRNA expressions of GSK-3βand PKA in brain tissues(P<0.05).And their mRNA expression of PP2A significantly increased(P<0.05).There was a significant decrease in the protein expression of GSK-3βin the cortical region of rat brain tissues in the medium-dose TSG group(P<0.05),as well as in the protein expression of GSK-3βin hippocampus of the medium-dose TSG group and the high-dose TSG group(P<0.01).The medium-dose TSG group and high-dose TSG group had significantly decreased protein expressions of PKA(P<0.05).Each TSG group had significantly decreased expression of protein Tau in brain tissues of rats(P<0.05or P<0.01),but significantly increased protein expression of PP2A(P<0.05or P<0.01).Conclusion TSG can regulate the activity of GSK-3β/PP2Aand interfere with Tau phosphorylation in the Aβ_(25-35)-induced dementia-like rat model,thus having an anti-dementia effect.The mechanism may be related to down-regulation of GSK-3βand PKA expressions and up-regulation of PP2Aexpression.