β-catenin RNA干扰对黄精丸治疗学习记忆障碍小鼠的信号通路机制的影响

Effect of β-catenin RNAi Interference on Signal Mechanism of Huangjingwan in Treating Learning and Memory Impairment Mice

目的:用侧脑室β-连环蛋白(β-catenin)RNA干扰(RNAi)黄精丸治疗D-半乳糖联合东莨菪碱模拟的阿尔茨海默病(AD)小鼠,以探讨黄精丸防治AD的神经保护信号分子机制。方法:81只雄性昆明种小鼠随机分成正常组,假手术组,AD模型组,多奈哌齐组,黄精丸+scrambled组(即β-catenin RNA干扰组),黄精丸+RNAi组,黄精丸组,多奈哌齐组和黄精丸组每组小鼠8只,其余各组每组13只。连续5周采用D-半乳糖联合东莨菪碱注射法给后5组的各小鼠制作AD模型。造模后第4周第1天,给黄精丸+RNAi组各小鼠右侧脑室一次性注射聚乙烯亚胺(PEI-LMW)/β-catenin siRNAs纳米络合物0.75μL以脑内β-catenin RNA干扰处理,黄精丸+scrambled组各小鼠右侧脑室一次性注射络合复合物0.75μL以作β-catenin干扰对照,假手术组各小鼠侧脑室内注射生理盐水0.75μL。侧脑室注射2周后,经确认小鼠β-catenin RNAi成功,再给多奈哌齐组小鼠灌胃多奈哌齐(6.5×10^(-4)g·kg^(-1)),给黄精丸+scrambled组,黄精丸+RNAi组,黄精丸组各小鼠灌胃黄精丸(2.5 g·kg^(-1)),持续4周。灌胃结束后,采用跳台实验评价各小鼠的学习记忆能力差异;尼氏染色计数各组小鼠大脑皮层S1Tr区及海马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)检测各组小鼠大脑Wnt1,蓬松蛋白极性片段2(DVL2),糖原合成酶激酶3β(GSK-3β),β-catenin,细胞周期蛋白D1(CyclinD1)mRNA表达水平;蛋白免疫印迹法(Western blot)检测各组小鼠大脑Wnt1,DVL2,GSK-3β,β-catenin,CyclinD1蛋白表达水平。结果:AD小鼠侧脑室内β-catenin RNAi可明显抑制其脑内β-catenin表达,可成功实现目的基因沉默。与正常组比较,AD模型组小鼠学习与记忆成绩显著下降,跳台的错误次数增多(P<0.01),大脑皮层S1Tr区及海马CA1,CA3区神经元神经元数量显著减少(P<0.01),大脑Wnt1,DVL2,β-catenin,CyclinD1mRNA及蛋白表达水平均显著下降(P<0.01),GSK-3βmRNA及蛋白表达水平显著升高(P<0.01)。与AD模型组比较,黄精丸组小鼠的学习与记忆成绩均显著升高,跳台错误次数均显著降低,大脑皮层S1Tr区及海马CA1,CA3区神经元数量显著升高,大脑Wnt1,DVL2,β-catenin,CyclinD1mRNA及蛋白表达水平均显著升高,GSK-3βmRNA及蛋白表达水平显著降低(P<0.01)。与黄精丸组比较,黄精丸+RNAi组小鼠的学习与记忆成绩均下降,跳台错误次数显著升高(P<0.01),大脑皮层S1Tr区及海马CA1,CA3区神经元数量显著降低(P<0.01),大脑Wnt1,DVL2,GSK-3βmRNA及蛋白表达水平无明显变化,β-catenin,CyclinD1mRNA及蛋白表达水平显著降低(P<0.01)。结论:黄精丸可通过激活Wnt/β-catenin信号通路转导发挥治疗AD作用。

Objective: To treat mice with Alzheimer’s disease(AD)with β-catenin RNA interference(RNAi)Huangjingwan(HW),so as to explore the neuroprotective signal mechanism of its prevention and treatment of AD. Method:A total of 81 male Kunming mice were randomly divided into normal control group,sham model control group,AD model group,Donepezil group,HW+scrambled group,HW+RNAi group,HW group,with 8 mice in each of donepezil group and HW group,and 13 mice in each of other groups. The AD models were established through injection with D-galactose and scopolamine in the last 5 groups for 5 consecutive weeks. On the 1 stday of the 4 thweek after modeling,0.75 μL PEI-LMW/β-catenin siRNAs nanocomplex was injected into the right lateral ventricle of each mouse in for one time to treat with β-catenin RNAi in mice brains of the HW+RNAi group. The 0.75 μL complex was injected into the right lateral ventricle of each mouse for one time as for β-catenin interference control of the HW+scrambled group. The 0.75 μL normal saline was injected into the right lateral ventricle of each mouse in one time of the sham control group. Two weeks after intracerebroventricular injection, β-catenin RNAi was confirmed to be successful, and Donepezil(6.5×10^(-4) g·kg^(-1)) was intragastrically administered to each mouse of donepezil group. HW(2.5 g·kg^(-1))was intragastrically administered to each mouse of HW group,HW+RNAi group and HW+scrambled group. Normal saline(0.5 mL·d-1)was intragastrically administered to each mouse of the sham control group. All gastric perfusion lasted for 4 weeks. At the end of gavage,the difference in learning and memory ability of mice was evaluated by platform jumping test. Nissl staining was used to count the number of neurons in s1 Tr area of cerebral cortex and CA1 and CA3 areas of hippocampus of each mouse in each group. The mRNA expressions of Wnt1,DVL2,GSK-3β,β-catenin and CyclinD1 in mice brain of each group were detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR). Western blot was used to detect the expressions of Wnt1,DVL2,GSK-3β,β-catenin and CyclinD1 in mice brain of each group. Result: The expression of β-catenin could be significantly inhibited through the injection with PEI-LMW/β-catenin siRNAs nano-complex into the lateral ventricle of AD mice,and nearly no β-catenin expression could be detected,which successfully achieved gene silencing. Compared with the normal control group,mice in AD model group showed that the learning and memory performance decreased significantly,the number of jumping errors increased(P<0.01),the number of neurons in S1 Tr area of cerebral cortex and CA1,CA3 areas of hippocampus decreased significantly(P<0.01),the mRNA and protein expressions of Wnt1,DVL2,β-catenin,CyclinD1 in brain decreased significantly(P<0.01),while the mRNA and protein expressions of GSK-3β increased significantly(P<0.01). Compared with the AD model group,mice in HW group showed that the learning and memory performance increased significantly,the number of jumping errors decreased,the number of neurons in S1 Tr area of cerebral cortex and CA1,CA3 areas of hippocampus increased significantly,the mRNA and protein expressions of Wnt1,DVL2,β-catenin,CyclinD1 in brain increased significantly,while the mRNA and protein expression of GSK-3β decreased significantly(P<0.01). Compared with the HW group,mice in HW+RNAi group showed that the learning and memory performance decreased significantly,the number of jumping errors increased significantly(P<0.01),the number of neurons in S1 Tr area of cerebral cortex and CA1,CA3 areas of hippocampus decreased significantly(P<0.01), there was no significant change in mRNA and protein expressions of Wnt1,DVL2,GSK-3β in the brain,and the mRNA and protein expressions of β-catenin,CyclinD1 decreased significantly(P<0.01). Conclusion: HW can treat and prevent AD by activating Wnt/β-catenin signal pathway.