三补方对自然衰老小鼠认知功能及脂质过氧化损伤的影响

Effects of Sanbu Decoction on cognitive function and lipid peroxidation damage in naturally aging mice

目的观察三补方对自然衰老小鼠认知功能的影响,探讨潜在病理因素和三补方(Sanbu Decoction,SBD)的作用及其可能作用机制。方法18只18月龄自然衰老健康SPF级野生型C57BL/6小鼠,雌雄各半,随机分为衰老组(WT-Aged)、多奈哌齐组(WT-Donepezil)、三补方组(WT-SBD),另选6只3月龄小鼠为青年对照组(WT-Young)。WT-Young小鼠不灌胃,WT-Aged小鼠用蒸馏水灌胃,其余用相应药物处理,灌胃45 d。灌胃结束前6日采用水迷宫实验,记录潜伏期时间、穿越平台次数和平台所在象限游泳路程。用HE染色观察海马神经元形态变化,免疫组织化学法测定小鼠脑海马组织Tau、p-Tau、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经上皮干细胞蛋白(neuroepithelial stem cell protein,Nestin)表达水平,检测试剂盒检测超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性和丙二醛(malondialdehyde,MDA)含量。结果与WT-Young相比,第2日起WT-Aged逃逸潜伏期延长(P<0.05),穿越平台次数和平台所在象限经过的路程减少(P<0.05);与WT-Aged相比,WT-Donepezil第3~5日和WT-SDB第4~5日逃逸潜伏期缩短(P<0.05),穿越平台次数和在平台所在象限经过的路程增多(P<0.05)。WT-Aged小鼠出现海马神经元排列紊乱和大面积的神经元坏死,细胞质嗜酸性增强,细胞核固缩、溶解、消失。与WT-Aged小鼠相比,WT-Donepezil和WT-SDB小鼠神经元损伤减少,排列较为紧密,退化固缩的细胞数量则明显减少,少部分细胞可见核固缩及细胞浆嗜酸性变。与WT-Young相比,WT-Aged Tau、p-Tau和GFAP表达增多(P<0.05),Nestin表达减少(P<0.05),SOD、GSH-Px活性降低(P<0.05),MDA含量升高(P<0.05)。与WT-Aged相比,WT-Donepezil和WT-SDB p-Tau、GFAP表达减少(P<0.05),WT-SBD SOD、GSH-Px活性升高(P<0.05),MDA含量降低(P<0.05)。结论三补方可以降低自然衰老小鼠脑海马组织p-Tau表达,减弱星形胶质细胞活化水平,降低脂质过氧化反应,发挥脑保护抗衰老作用,提高老年小鼠学习记忆能力,改善衰老小鼠的衰老状态,延缓衰老进程。

Objective To observe the effects of Sanbu Decoction(SBD)on the cognitive function of naturally aging mice,and to explore the involved potential pathological factors,as well as the action of SBD and its possible mechanism.Methods Eighteen healthy SPF wild-type C57BL/6 naturally aging mice,half female and half male,aged 18 months,were randomized into aging group(WT-Aged),donepezil group(WT-Donepezil),SBD group(WT-SBD),and another six mice aged three months were selected as the young control group(WT-Young).WT-Young group was not treated.WT-Aged group were treated with distilled water,and the rest groups with corresponding drugs,by gavage for 45 days.During the six days before the end of gavage,Morris water maze test was performed,and the escape latency,platform crossing times,and the swimming distance in the platform quadrant were recorded.HE staining was carried out to observe the morphologic changes of hippocampal neurons of the mice.Immunohistochemistry was applied to measuring the expression levels of Tau,p-Tau,glial fibrillary acidic protein(GFAP),and neuroepithelial stem cell protein(Nestin)in the hippocampal tissue of the mice.The detection kits were used to check the activity of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)and the content of malondialdehyde(MDA).Results Compared with WT-Young group,the escape latency of WT-Aged group was prolonged from the 2nd day(P<0.05),and the platform crossing times and the swimming distance in the platform quadrant were reduced(P<0.05);compared with WT-Aged group,the escape latency of WT-Donepezil group on the 3rd to 5th day and WT-SBD group on the 4th to 5th day was shortened(P<0.05),and platform crossing times and the swimming distance in the platform quadrant were increased(P<0.05).The hippocampal neurons in WT-Aged group showed disordered arrangement and large areas of necrosis,enhanced acidophilia in the cytoplasm,and pyknosis,dissolution,and disappearance of the nuclei.Compared with WT-Aged group,the hippocampal neurons in WT-Donepezil and WT-SBD groups showed reduced damage and compact arrangement,and the degenerated and pyknotic cells were significantly decreased,with a small minority showing karyopyknosis and cytoplasmic acidophilic change.Compared with WT-Young group,the expressions of Tau,p-Tau,and GFAP in WT-Aged group increased(P<0.05),the expression of Nestin and the activity of SOD and GSH-Px decreased(P<0.05),and the content of MDA was elevated(P<0.05);compared with WT-Aged group,the expressions of p-Tau and GFAP decreased in WT-Donepezil and WT-SBD groups(P<0.05),and the activity of SOD and GSH-Px in WT-SBD group increased(P<0.05)while the content of MDA was lowered(P<0.05).Conclusion SBD can reduce the expression of p-Tau in the hippocampus of naturally aging mice,weaken the activation level of astrocytes,and reduce lipid peroxidation,which can improve the learning and memory abilities of senile mice,alleviate their aging state,and delay the aging process,thus playing the roles of brain protection and anti-aging.