黄精丸抑制D-半乳糖和冈田酸所致学习记忆障碍小鼠海马神经元tau蛋白过磷酸化的作用机制

Mechanism of Huangjingwan in Inhibiting Tau Hyperphosphorylation in Hippocampal Neurons of Mice with Alzheimer’s Disease Induced by D-galactose and Okadaic Acid Resulting in Learning and Memory Disorders

目的:探讨黄精丸对D-半乳糖和冈田酸所致学习记忆障碍造模的阿尔茨海默病(AD)小鼠大脑海马神经元糖原合成酶激酶3β(GSK-3β),蛋白磷酸酶2A(PP2A)活性的影响及其抑制tau蛋白过磷酸化机制。方法:采用小鼠颈背部皮下注射1.0%D-半乳糖液(0.14 g·kg^(-1)·d^(-1),连续4周)后,再右侧脑室一次性注射(75 ng)冈田酸2μL,制作小鼠AD模型,经Morris水迷宫检测挑选造模成功的AD小鼠,再随机分为AD模型组,美金刚组(1.3×10^(-3)g·kg^(-1)·d^(-1)),黄精丸组(2.5 g·kg^(-1)·d^(-1)),另设假手术组和正常组;同时点给假手术组的小鼠右侧脑室一次性注射生理盐水2μL处置作造模对照。造模2周后给两实验药物组小鼠灌胃相应剂量实验药物,持续4周。另外,AD造模2周后,同时给假手术组及AD模型组小鼠每天灌胃等量生理盐水,持续4周;正常组小鼠仅日常饲养。灌胃结束后,采用旷场实验及跳台实验评价各小鼠的探索活动能力、焦虑水平及学习记忆能力差异;尼氏染色检测各组小鼠海马CA1,CA3区神经元数量差异;实时荧光定量聚合酶链式反应(Real-time PCR)检测各组小鼠海马GSK-3β,PP2A mRNA表达变化,蛋白免疫印迹法(Western blot)检测各组小鼠海马GSK-3β,PP2A,磷酸化tau(p-tau)与总tau(t-tau)蛋白的表达变化。结果:与正常组比较,AD模型组小鼠自发活动能力与探索行为能力低、焦虑水平高,表现出较明显的痴呆状态,少动且易呆卧躲藏,学习反应时间要长,学习记忆错误次数明显增多,海马CA1与CA3区神经元数量均减少,PP2A mRNA及蛋白表达显著下降,GSK-3βmRNA及其蛋白表达,p-tau蛋白水平及p-tau/t-au值均显著升高(P<0.01),t-tau蛋白表达水平有所降低但差异无统计学意义。与AD模型组比较,黄精丸组小鼠自发活动能力与探索行为能力强、焦虑水平低、学习与记忆成绩明显提高,海马CA1与CA3区神经元数量增多,PP2A mRNA及蛋白表达显著升高,GSK-3βmRNA及蛋白表达,p-tau蛋白水平及p-tau/t-tau值均显著降低(P<0.01),但t-tau蛋白表达水平差异无统计学意义。结论:黄精丸可抑制AD小鼠海马神经元tau蛋白过磷酸化,恢复tau蛋白功能,保护海马神经,进而发挥抗AD作用,其机制可能与调控海马神经元GSK-3β与PP2A活性平衡有关。

Objective: To investigate the effect of Huangjingwan(HW)on the activities of glycogen synthase kinase-3β(GSK-3β),protein phosphatase 2 A(PP2 A)and the mechanism in inhibiting tau protein hyperphosphorylation in the hippocampal neurons of mice with Alzheimer’s disease. Method: After subcutaneous injection with 1.0% D-galactose(0.14 g·kg^(-1)·d^(-1))into the back and neck of mice for 4 weeks,the right ventricle of mice was injected with 2 μL(75 ng)of okadaic acid for one time to make AD model,and the successfully modeled AD mice were selected by Morris water maze. Then,the selected AD mice were randomly divided into AD model group, memantine group(1.3×10^(-3) g·kg^(-1)·d^(-1)) and HW group(2.5 g·kg^(-1)·d^(-1)). In addition,the sham model control group and the normal control group were set up. At the same time,2 μL normal saline was injected into the right ventricle of mouse in the sham model control group for modeling control. Two weeks after modeling,the mice in the two experimental drug groups were given the corresponding dose of the experimental drug by gavage for 4 weeks. In addition,after 2 weeks of AD modeling,mice in control group and AD model group were intragastrically administrated with the same amount of normal saline daily for 4 weeks. The mice in normal control group were only given daily feed. At the end of gavage,all the mice were tested by the open field experiment and jumping platform experiment to evaluate the differences in exploratory activity ability,anxiety level and learning and memory ability. The number of neurons in CA1 and CA3 areas of hippocampus in all the mice was detected by Nissl staining. Quantitative real-time polymerase chain reaction(Real-time PCR)was used to detect m RNA expressions of GSK-3β and PP2 A in hippocampus of mice in each group. Protein expressions of GSK-3β,PP2 A,phosphorylated tau(p-tau)and total tau protein(t-tau)in hippocampus of mice in each group were detected by Western blot. Result: Compared with the normal control group,mice in AD model group showed an obvious dementia state,which was characterized by a lower spontaneous activity,lower exploration behavior ability,higher anxiety level,less movement and easier to stay and hide,longer learning response time,significantly increased number of learning and memory errors,and decreased numbers of hippocampal neuron in CA1 and CA3 areas,and reduced m RNA and protein expressions of PP2 A,m RNA and protein expressions of GSK-3β,p-tau protein and the ratio of p-tau/t-tau were all increased significantly(P<0.01), while expression of t-tau protein was decreased, with no significant difference.Compared with the AD model group,mice in the HW group showed a higher spontaneous activity,higher exploration ability, lower anxiety level, higher learning and memory performance, and the numbers of hippocampal neuron in CA1 and CA3 areas increased,while m RNA and protein expressions of PP2 A increased,and the m RNA and protein expressions of GSK-3β,the expression of p-tau protein and the ratio of p-tau/t-tau were all decreased significantly(P<0.01),but with no significant difference in the protein expression of t-tau.Conclusion: HW can inhibit tau hyperphosphorylation in hippocampal neurons of AD mice,restore tau protein function,protect hippocampal neurons,and exert an anti-AD effect,which may be related to the regulatory mechanism in the activity balance between GSK-3β and PP2 A in hippocampal neurons.