黄芪多糖对阿尔茨海默病大鼠神经细胞活性、认知功能及Caspase-9表达水平的影响

Effect of Astragalus polysaccharide on neuronal activity, cognitive function and Caspase-9 expression in Alzheimer’s disease rats

目的探讨黄芪多糖(Astragalus polysacharin, APS)对阿尔茨海默病(Alzheimer’s disease, AD)大鼠神经细胞活性、认知功能及天冬氨酸特异性半胱氨酸蛋白酶(Cysteinyl aspartate specific protease, Caspase)-9表达水平的影响。方法 36只无特定病原体(Specific pathogen free, SPF)级美国斯泼累格·多雷(Sprague Dawley)雌雄各半的大鼠,按照随机数字表分为6组,正常组、AD组、药物对照组、干预A组、干预B组及干预C组;除正常组外,其余大鼠建立AD大鼠模型;建模后药物对照组采用0.5 g/kg的吡拉西坦灌胃,干预A组、干预B组及干预C祖均采用0.2、0.4及0.8 g/kg的黄芪多糖(Astragalus polysacharin, APS)灌胃,正常组及AD组灌胃等剂量的生理盐水,均1次/d,灌胃时间连续60 d。采用Morris水迷宫实验观察认知功能;苏木精-伊红(Hematoxy lin-Eosin, HE)染色观察海马组织病理学表现;末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法[Terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling, TUNEL]法检测神经细胞凋亡率;免疫印迹及实时定量聚合酶链反应(Quantitatie real time polymerase chain reaction, QRT-PCR)技术分别检测细胞色素C(Cytochrome c, Cyt-c),Caspase-3及Caspase-9蛋白及信使核糖核酸(Messenger RNA,mRNA)相对表达水平。结果与正常组比较,AD组大鼠灌胃后第1~5 d的逃避潜伏期均延长(P<0.05);与AD组比较,干预A组、干预B组、干预C组逃避潜伏期均缩短(P<0.05),药物对照组逃避潜伏期与干预C组比较无明显差异(P>0.05)。与正常组比较,AD组大鼠游泳距离增加(P<0.05);与AD组比较,干预A组、干预B组及干预C组大鼠游泳距离均减少(P<0.05),干预C组与药物对照组相似(P>0.05)。正常组海马结构完成,神经元排列紧密,细胞核清晰且无空泡;AD组大鼠海马组织结构紊乱,神经元数目减少,细胞核深染,细胞膜收缩及部分消失;APS干预组及药物对照组神经元排列较AD组整齐有序,肿胀程度减轻,细胞核较清晰。各组大鼠海马组织神经细胞凋亡率比较有明显差异(F=134.900,P<0.001);与正常组比较,AD组神经细胞凋亡率升高(P<0.05);与AD组比较,干预A组、干预B组及干预C组神经细胞凋亡率降低(P<0.05),药物对照组与干预C组神经细胞凋亡率相似(P>0.05)。与正常组比较,AD组海马组织Cyt-C,Caspase-3及Caspase-9蛋白及mRNA相对表达水平上调(P<0.05);与AD组比较,不同水平干预组海马组织Cyt-C,Caspase-3及Caspase-9蛋白及mRNA相对表达水平下调(P<0.05),药物对照组与干预C组上述蛋白及mRNA相对表达水平相似(P>0.05)。结论黄芪多糖能够改善AD大鼠认知功能,减轻海马组织病理损伤,抑制神经元凋亡且呈现水平依赖性,其机制可能与抑制Cyt-C及caspase-3/9信号通路有关。

Objective To explore the effect of Astragalus polysacharin(APS) on neuronal activity, cognitive function and cysteinyl aspartate specific protease-9(caspase-9) expressionin Alzheimer’s disease(AD) rats.Methods 36 specific pathogen-free(SPF) Sprague Dawley male and female rats were randomly divided into 6 groups including normal group, AD group, drug control group, intervention group A, intervention group B and intervention group C. Except the normal group, AD rat model was established in other groups. After modeling, the drug control group was gavaged with 0.5 g/kg piracetam. The intervention group A, intervention group B and intervention group C were gavaged with 0.2, 0.4 and 0.8 g/kg APS, respectively. The normal group and the AD group were gavaged with equal dose of normal salineonce a day. The gavage was performed continuously for 60 days. Morris water maze test was used to evaluate cognitive function. Hematoxy lin-eosin(HE) staining was performed to observe the histomathological manifestations of the hippocampus. Terminal dexynucleotidyl transferase mediated DUTP Nick end labeling(TUNEL) method was used to detect the apoptosis rate of nerve cells. Western blot and quantitative real-time PCR(QRT-PCR) technique were performed to detect protein and mRNA expression levels of cytochrome C(Cyt-C), caspase-3 and caspase-9, respectively.Results Compared with the normal group, rats in the AD group had a longer escape latency from 1 to 5 days after gavage(P<0.05). Compared with the AD group, the escape latency of the intervention group A, the intervention group B, and the intervention group C was all shortened(P<0.05). Additionally, the escape latency of the drug control group was not significantly different from that of the intervention group C(P>0.05). Compared with the normal group, the swimming distance of rats in the AD group was significantly increased(P<0.05). Compared with the AD group, the swimming distance of rats in the intervention group A, the intervention B group and the intervention C group was all reduced(P<0.05), and the swimming distance was similar between the intervention group C and the drug control group(P>0.05). The hippocampus structure of the normal group was complete, the neurons were tightly arranged and the nucleus was clear without empty bubbles. The AD group has disordered hippocampal tissue structure, decreased number of neurons, dark-stained nuclei, and shrinkage and partial disappearance of cell membranes. In the APS intervention group and the drug control group, the arrangement of neurons was more neat and orderly than that in the AD group, the swelling was reduced, and the nucleus was clear. The neuronal apoptosis rate in the hippocampus of the rats was relatively different among different groups(F=134.900, P<0.001). Compared with the normal group, the neuronal apoptosis rate in the AD group was up-regulated(P<0.05). Compared with the AD group, the apoptosis rate of nerve cells in intervention group A, intervention group B and intervention group C was significantly decreased(P<0.05). The apoptosis rate in the drug control group was similar to that in intervention group C(P>0.05). Compared with the normal group, the protein and mRNA expression of hippocampal Cyt-C, caspase-3 and caspase-9 in the AD group was up-regulated(P<0.05). Compared with the AD group, the protein and mRNA expression levels of Cyt-C, caspase-3 and caspase-9 were down-regulated in the hippocampus in the intervention groups with various concentrations(P<0.05). The above-mentioned protein and mRNA expression levels in the drug control group and intervention group C were similar(P>0.05).Conclusion Astragalus polysaccharide could improve the cognitive function of AD rats, reduce the pathological damage of hippocampus, and inhibit neuronal apoptosis in a concentration-dependent manner. The underlying mechanisms might be related to the inhibition of Cyt-C and caspase-3/9 signaling pathway.