摘要
目的观察归脾汤对甲基丙二酸血症(methylmalonic acidemia,MMA)模型大鼠学习记忆能力及大脑海马组织中p-AMPK、线粒体氧化应激损伤相关蛋白表达的影响,探讨其作用机制。方法出生5天的Wistar清洁级大鼠幼鼠随机选取分组(8只/组),即正常组、模型组、低剂量归脾汤组、中剂量归脾汤组、高剂量归脾汤组,皮下注射甲基丙二酸溶液建立MMA模型,采用Morris水迷宫实验观察各组大鼠行为学变化,透射电子显微镜观察大鼠海马组织CA1区神经元超微结构,酶联免疫吸附测定法(Enzyme-Linked Immunosorbent Assay,ELISA)检测海马组织超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)含量,Western blotting法检测p-AMPK及HO-1、NQO1、Caspase-3的表达水平。结果Morris水迷宫实验结果表明,与正常组相比,模型组大鼠穿越平台次数、穿越目标象限时间占比均明显减少(P<0.05,P<0.01);与模型组相比,高剂量组穿越目标象限时间占明显增多(P<0.01),低、中、高剂量组穿越平台次数均明显增多(P<0.05、P<0.05、P<0.01)。透射电子显微镜观察各组大鼠海马组织CA1区,正常组显示神经元细胞线粒体、突触后膜无明显异常;与空白对照组相比,模型组细胞形态破坏,细胞异染色质变粗大、凝结成块,线粒体、粗面内质网、高尔基体水肿,线粒体堆积、小泡堆积突触后膜晕染、模糊,溶酶体增多;与模型组相比,高剂量组细胞形态正常,有较完整的线粒体,且无线粒体堆积。ELISA检测表明,与正常组相比,模型组海马组织中SOD含量显著降低(P<0.05),MDA含量显著升高(P<0.01)。与模型组相比,低剂量组海马组织中MDA含量显著降低(P=0.023,P<0.05),低剂量、高剂量归脾汤组海马组织中SOD含量均显著升高(P<0.05,P<0.01),低剂量组MDA含量显著降低(P<0.05)。Western blotting检测提示,与正常组相比,模型组p-AMPK、HO-1、NQO1蛋白表达水平均降低(P<0.01),Caspase-3蛋白表达水平升高(P<0.01);与模型组相比,低剂量组、中剂量组和高剂量组p-AMPK蛋白表达水平升高(P<0.05),高剂量组HO-1、NQO1蛋白表达水平均升高(P<0.05),而Caspase-3蛋白表达水平降低(P<0.01)。结论归脾汤可以改善MMA模型大鼠的学习记忆能力,可能是通过调节AMPK减轻海马组织线粒体氧化应激损伤发挥治疗作用。
Objective To observe the effects of Guipi Decoction(归脾汤)on the learning and memory abilities as well as the expressions of p-AMPK and mitochondrial oxidative stress-related proteins in the hippocampus of rats with methylmalonic acidemia(MMA)model and explore its mechanism of action.Method Wistar clean grade rats born for 5 days were randomly divided into groups(8 rats/group):normal group,model group,Guipi Decoction low dose group,Guipi Decoction medium dose group,Guipi Decoction high dose group.The MMA model was established by subcutaneous injection of methylmalonic acid solution.Morris water maze experiment was used to observe the behavioral changes of rats in each group.The transmission electron microscopy was used to observe the ultrastructure of neurons in the CA1 area of rat hippocampal tissue.Enzyme-Linked Immunosorbent Assay(ELISA)was used to detect the contents of superoxide dismutase(SOD)and malondialdehyde(MDA)in hippocampal tissue,and Western blotting was used to detect the expression levels of p-AMPK,HO-1,NQO1 and caspase-3.Result The Morris water maze experiment showed that compared with that of the normal group,the times of crossing the platform and the time of crossing the target quadrant of the model group were significantly reduced(P<0.05,P<0.01).Compared with that of the model group,the time of crossing the target quadrant in the high dose group increased significantly(P<0.01),while the times of crossing the platform of the low,medium and high dose groups increased significantly(P<0.05,P<0.05,P<0.01).Transmission electron microscopy was used to observe the CA1 region of hippocampal tissue in each group.The normal group showed no significant abnormalities in neuronal cell mitochondria or postsynaptic membranes.Compared with that of the normal group,the cell morphology of the model group was disrupted,and the heterochromatin of the cells became thicker and clumped,with edema in mitochondria,rough endoplasmic reticulum and Golgi apparatus.Mitochondria and vesicles accumulated,and the postsynaptic membrane was stained and blurred,with an increase in lysosomes.Compared with the model group,the high dose group had normal cell morphology,more complete mitochondria and no accumulation of mitochondria.ELISA detection showed that compared with that of the blank group,the SOD content in the hippocampal tissue of the model group was significantly reduced(P<0.05),while the MDA content was significantly increased(P<0.01).Compared with that of the model group,the MDA content in the hippocampus tissue of the low dose group was significantly reduced(P=0.023,P<0.05),and the SOD content in the hippocampal tissue of both low dose and high dose groups significantly increased(P<0.05,P<0.01),while the MDA content in the low dose group significantly decreased(P<0.05).Western blotting analysis showed that compared with the normal group,the protein expression levels of p-AMPK,HO-1 and NQO1 in the model group decreased(P<0.01),while the protein expression level of Caspase-3 increased(P<0.01).Compared with those of the model group,the expression levels of p-AMPK protein increased in the low,medium and high dose groups(P<0.05),while the expression levels of HO-1 and NQO1 protein increased in the high dose group(P<0.05),while the expression levels of Caspase-3 protein decreased(P<0.01).Conclusion Guipi Decoction can improve the learning and memory abilities of MMA model rats,possibly by regulating AMPK to alleviate mitochondrial oxidative stress damage in hippocampal tissue and exert therapeutic effects.
作者
陆相朋
郑宏
郑亚蓓
张诺楠
白皇凰
李璐瑶
李晓孟
LU Xiangpeng;ZHENG Hong;ZHENG Yabei;ZHANG Nuonan;BAI Huanghuang;LI Luyao;LI Xiaomeng(The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,Henan,China;School of Pediatrics,Henan University of Traditional Chinese Medicine,Zhengzhou 450000,Henan,China;Shenzhen Hospital of Traditional Chinese Medicine,Shenzhen 518033,Guangdong,China)
出处
《辽宁中医杂志》
北大核心
2026年第2期186-192,共7页
Liaoning Journal of Traditional Chinese Medicine
基金
国家自然科学基金面上项目(82274579)
国家自然科学基金青年科学基金项目(82104929)
国家中医临床研究基地科研专项(2021JDZY027)
河南省科技厅科技攻关项目(212102310364)。
关键词
归脾汤
甲基丙二酸血症
认知障碍
AMPK
线粒体氧化应激
Guipi Decoction(归脾汤)
methylmalonic acidemia
cognitive impairment
AMPK
mitochondrial oxidative stress
