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还脑益聪方提取物对APP/PS1双转基因痴呆模型小鼠APH-1及PEN-2基因调控通路的影响
【作者】 王琦；
【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2016， 博士
【摘要】 目的观察中药小复方还脑益聪方(Huannao Yicongfang, HNYCF)提取物对APP/PS1双转基因小鼠痴呆模型Y分泌酶APH-1、PEN-2相关通路的干预作用,探讨还脑益聪方治疗老年性痴呆(AD)可能的作用机制。方法选取3月龄APP/PS1双转基因痴呆模型小鼠60只随机分为模型组、西药组、还脑益聪方低剂量组(HNYCF小组)、还脑益聪方高剂量组(HNYCF大组),每组15只,另取3月龄相同遗传背景的C57BL/6J小鼠15只作为正常对照组,各组灌胃给药,每日一次,连续灌胃3个月,正常组和模型组灌胃等体积蒸馏水,西药组灌胃盐酸多奈哌齐(0.65mg/kg)水溶液,中药各剂量组分别按照5.46g生药/kg、10.92g生药/kg灌胃给药,给药结束后进行水迷宫实验观察不同组别学习记忆和认知功能的变化,ELISA法检测各组小鼠海马组织淀粉样前体蛋白(APP)和β淀粉样蛋白(Aβ)的含量；双荧光素酶方法检测各组小鼠Y分泌酶活性；RT-PCR、Western-blot法分别检测各组小鼠海马组织中前咽缺陷蛋白-1(APH-1)、缺氧诱导因子1(HIF-1)、早老蛋白增强子-2(PEN-2)；环磷酸腺苷应答元件结合蛋白(CREB)等相关基因mRNA及蛋白的表达。结果第一部分：Morris水迷宫定位航行实验显示,与正常组比较,模型组和其余各组航行总时间均显著延长(P<0.01),模型组、HNYCF药小组小鼠第四象限路程显著延长(P<0.05或P<0.01)；经3个月用药之后,与模型组比较,各用药组小鼠航行总时间显著缩短(P<0.01),西药组和HNYCF大组显著缩短(P<0.05)；各用药组之间比较无显著性差异(P>0.05)。空间探索实验显示,与正常组比较,模型组、HNYCF组小鼠的航行的平均速度、第一次穿台时间均有显著性差异(P<0.01)；经3个月用药之后,与模型组比较,西药组小鼠的航行的平均速度、第一次穿台时间均有显著性差异(P<0.01),中药大组的第一次穿台次数有统计学意义(P<0.05)；与西药组比较,HNYCF小组航行的平均速度有显著差异(P<0.01)。第二部分：ELISA结果提示,与正常组比较,模型组小鼠海马CA1区的APP、Aβ1-40及Aβ1-42表达均显著增加(P<0.01),HNYCF小组Aβ1-42表达也增加(P<0.01)；经3个月用药之后,与模型组比较,西药组APP、Aβ1-40及Aβ1-42含量均减少(P<0.05)；且HNYCF大组的APP、Aβ1-42含量减少(P<0.05),与西药组比较,HNYCF小组Aβ1-42含量有显著差异(P<0.05)。第三部分：双荧光素酶报告基因系统结果显示,与正常组比较,模型组显著增高(P<0.01)；经3个月用药之后,与模型组比较,西药组的Y分泌酶活性无显著性差异(P>0.05),中药大组的Y分泌酶活性显著降低(P<0.05)；其余各组无差异(P>0.05)。第四部分：Real-time PCR提示,与正常组比较,模型组及各用药组小鼠的APH-1a mRNA=HIF-1a mRNA表达均显著升高(P<0.05或P<0.01),模型组小鼠的PEN-2mRNA、 CREB mRNA表达显著升高(P<0.01),西药组小鼠的PEN-2 mRNA表达表达显著升高(P<0.01)；经3个月用药之后,与模型组比较,HNYCF小组及大组PEN-2 mRNA、CREB mRNA表达显著降低(P<0.05或P<0.01)；各用药组小鼠的表达无显著性差异(P>0.05)。第五部分：Western-blot提示：与正常组比较,模型组及各用药组小鼠的APH-1a、 HIF-1a蛋白表达均显著升高(P<0.05或P<0.01),模型组小鼠的PEN-2、CREB蛋白表达显著升高(P<0.01),西药组小鼠的PEN-2蛋白表达显著升高(P<0.05)；经3个月用药之后,与模型组比较,HNYCF小组及大组PEN-2、CREB蛋白表达显著降低(P<0.05或P<0.01)；各用药组小鼠的蛋白表达无显著性差异(P>0.05)。结论还脑益聪方提取物可以明显改善APP/PS1双转基因小鼠学习记忆能力及空间认知能力,具有防治老年性痴呆的作用,其作用机制与抑制γ分泌酶活性,抑制CREB蛋白的活性进而下调PEN-2蛋白的表达这条调控通路来抑制γ分泌酶的活性,减少APP的裂解和Ap的产生相关。表明还脑益聪方提取物作为防治老年性痴呆的有效药物具有良好的研究开发前景。
【Abstract】 Objective:To observe the intervention effect of concise prescriptions of Chinese medicine (Huannao Yicongfang, HNYCF) extract on APH-1, PEN-2 regulatory pathway of y secretase in brain tissue of APP/PS1 double transgenic dementia mice model.To explore the mechanism of HNYCF extract and provide a scientific basis for development an effective drug for prevention and treatment of Alzheimer’s disease (AD).Methods:60 APP/PS1 double transgenic model mice (3 months old) were randomly divided into 5 groups, especially the normal group, the model group (n=15), Donepezil group (n=15), HNYCD low-dose group (n=15) and HNYCD high-dose group (n=15). Another 15 C57BL/6J mice of 3-month-old were selected into the normal control group. The normal group and the model group were given equal-volume distilled water, other groups were respectively given Donepezil (0.65mg/kg), Huannao Yicongfang low dosage extract (5.46g/kg) and Huannao Yicongfang high dosage extract (10.92g/kg) through gastric infusion. The administration was once a day, lasting for 3 months. The ability of memory and learning were tested by Morris water gaze, the content of amyloid precursor protein (APP) and β amyloid protein (Aβ) was detected by ELISA, the activity of gamma was detected by Dual Luciferase Reporter Gene Assay Kit. And the levels of APH-1 a, HIF-1α, PEN-2, CREB mRNA and protein in hippocampus were detected by RT-PCR and western-blot.Results:The first part:In the place navigation test of Morris water maze showed, compared with the normal control group, the time of sailing were prolonged obviously (P<0.01), and the swimming distance in the fourth quadrant was increased significantly in the model group and the low dosage of HNYCF group (P<0.01). After medication of 3 months, compared with the model group, the time of sailing were shortened obviously the western medicine control group and the control group (P<0.01), the swimming distance in the fourth quadrant was shortend significantly in the model group and the low dosage of HNYCF group (P<0.01). In the space probe trial of Morris water maze, compared with the normal control group, the average speed and the first cross-platform were significant difference between the control group and the low dosage of HNYCF group (P< 0.01), the first cross-platform were significant difference between the western medicine control group. After medication of 3 months, compared with the model group, the average speed and the first cross-platform were significant difference between the western medicine control group (P< 0.01), the first cross-platform were significant difference between the high dosage of HNYCF group (P< 0.05), there was significant difference in average speed between the western medicine control group and the low dosage of HNYCF group.The second part:ELISA showed that compared with the normal control group, the expression of APP, Aβ1-40 and A β1-42 all were increased significantly in hippocampal CA1 region of mice in the model group (P<0.01), the expression of Aβ1-42 was increased significantly in the low dosage of HNYCF group (P<0.01). After medication of 3 months, compared with the model group, the expression of APP, Aβ1-40 and A β1-42 were all reduced significantly in the western medicine control group (P<0.01), the expression of APP and Aβ1-42 in the low dosage of HNYCF group were reduced significantly (P<0.05). Compared with the western medicine control group, the expression of Aβ1-42 was increased significantly in the low dosage of HNYCF group (P<0.05).The third part:Dual Luciferase Reporter Gene Assay Kit showed that with the normal control group, the activity of gamma was increased significantly the model group (P<0.01). After medication of 3 months, compared with the model group, the activity of gamma was no significantly different in the western medicine control group (P>0.05), the activity of gamma was reduced in high dosage HNYCF group (P<0.05). There were no significantly different between the other groups (P>0.05).The fourth part:RT-PCR showed that compared with the normal control group, the expression of APH-la mRNA, HIF-1α mRNA all increased significantly in the model group and each medicine group (P<0.05 or P<0.01), the expression of PEN-2 mRNA, CREB mRNA increased significantly in the western medicine control group (P< 0.01), After medication of 3 months, compared with the model group, the expression of PEN-2 mRNA, CREB mRNA increased significantly in the low and high dosage HNYCF groups (P<0.01).The fifth part:RT-PCR showed that compared with the normal control group, the expression of APH-la, HIF-1α all increased significantly in the model group and each medicine group (P< 0.05 or P< 0.01), the expression of PEN-2, CREB increased significantly in the western medicine control group (P<0.05). After medication of 3 months, compared with the model group, the expression of PEN-2, CREB increased significantly in the low and high dosage HNYCF groups (P<0.01).Conclusion:HNYCF extract can improve learning and memory ability and spatial cognitive ability of APP/PS1 double transgenic mice obviously, its mechanism may be related to inhibit the activity of y secretase by down-regulating the activity of CREB protein and the expression of PEN-2 protein, and reduce the decomposition of APP and the production of A (3.This shows that HNYCF extract has promissing research and application as an effective drug for prevention and treatment of AD.