四君子汤对阿尔茨海默病大鼠行为学及海马神经元能量代谢功能的影响
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摘要
目的:观察阿尔茨海默病大鼠行为学及线粒体变化,探讨四君子汤治疗阿尔茨海默病大鼠行为学及对海马神经元线粒体能量代谢变化的机制。方法:将60只雄性SPF级大鼠随机分为正常组,模型组,四君子汤低、中、高剂量组(3. 24,6. 48,12. 56 g·kg~(-1)),双氢麦角碱组(0. 27 mg·kg~(-1)),每组10只。正常组不干预,饮食正常,其余大鼠以200 mg·kg~(-1)剂量向腹部注射D-半乳糖,每日注射1次,共计6周。采用Morris水迷宫实验检测大鼠认知功能;采用泰盟TM-Vision行为学实验系统观察大鼠行为学变化;采用透射电镜观察海马神经元线结构;采用比色法检测大鼠线粒体复合物CⅠ,CⅡ,CⅢ,CⅣ活性;采用蛋白免疫印迹法(Western blot)检测海马线粒体单磷酸腺苷活化蛋白激酶(AMPK)蛋白表达。结果:与正常组比较,模型组大鼠潜伏逃避期时间增加,穿越次数减少,运动距离增加,运动时间增多,中心停留时间增加(P <0. 05,P <0. 01),线粒体CⅡ,CⅢ,CⅣ活性下降(P <0. 05,P <0. 01),AMPK蛋白表达明显升高(P <0. 05,P <0. 01);与模型组比较,四君子汤中、高剂量组及双氢麦角碱组大鼠潜伏逃避期时间减少,穿越次数明显增加,运动距离明显减少,运动时间明显减少,中心停留时间明显减少(P <0. 05,P <0. 01);四君子汤中剂量组与模型组比较线粒体复合物CⅡ,CⅣ活性升高,AMPK蛋白表达下调(P <0. 05,P <0. 01),四君子汤高剂量组线粒体复合物CⅡ,CⅢ活性明显升高,AMPK蛋白表达明显下调(P <0. 05,P <0. 01)。结论:阿尔茨海默病大鼠行为学异常,可能与中枢海马能量代谢及线粒体功能下降有关,四君子汤有治疗作用。
Objective: To observe the changes of behavior and mitochondria in Alzheimer' s disease( AD) rats,and to explore the mechanism of Sijunzi Tang in the treatment of AD rats' behavior and the changes of mitochondrial energy metabolism in hippocampal neurons. Method: The 60 male SPF grade rats were randomly divided into normal group,model group,low,medium and high dose group of Sijunzi Tang( 3. 24,6. 48,12. 56 g·kg~(-1)),and dihydroergot group( 0. 27 mg·kg~(-1)),10 rats in each group. The normal group received no intervention and had a normal diet. The rest of the rats were injected with D-galactose to the abdomen at a dose of 200 mg·kg~(-1) once a day for 6 weeks. Morris water maze experiment was used to detect the cognitive function of rats. Tm-Vision behavioral experiment system was used to observe the behavioral changes of rats,and the hippocampal neuronal line structure was observed by transmission electron microscope. Mitochondrial complex colorimetry was adopted to detect rats C Ⅰ,C Ⅱ,C Ⅲ,C Ⅳ activity. Western blot was used to detect the expression of mitochondrial adenosine monophosphate-activated protein kinase( AMPK) protein in the hippocampus. Result: Compared with normal group,model group rats latent escape period time increasing,through fewer,movement distance,movement time increasing,center residence time increased( P < 0. 05,P <0. 01),mitochondria C Ⅱ,C Ⅲ,C Ⅳ activity decline( P < 0. 05,P < 0. 01),The AMPK protein expression increased significantly( P < 0. 05,P < 0. 01). Compared with model group,Sijunzi Tang medium and high dose group and dihydroergot decoction group showed decreased latency,increased crossing times,decreased exercise distance,decreased exercise time,and decreased center residence time( P < 0. 05,P < 0. 01). Compared with model group C Ⅱ,mitochondrial complex CⅣ activity increases,AMPK protein expression lowered( P < 0. 05,P < 0. 01),Sijunzi Tang high dose group of mitochondrial complex C Ⅱ,C Ⅲ activity increased significantly,AMPK protein expression significantly lowered( P < 0. 05,P < 0. 01). Conclusion: The abnormal behavior of AD rats may be related to the decrease of central hippocampal energy metabolism and mitochondrial function. Sijunzi Tang has therapeutic effect.
Objective: To observe the changes of behavior and mitochondria in Alzheimer' s disease( AD) rats,and to explore the mechanism of Sijunzi Tang in the treatment of AD rats' behavior and the changes of mitochondrial energy metabolism in hippocampal neurons. Method: The 60 male SPF grade rats were randomly divided into normal group,model group,low,medium and high dose group of Sijunzi Tang( 3. 24,6. 48,12. 56 g·kg~(-1)),and dihydroergot group( 0. 27 mg·kg~(-1)),10 rats in each group. The normal group received no intervention and had a normal diet. The rest of the rats were injected with D-galactose to the abdomen at a dose of 200 mg·kg~(-1) once a day for 6 weeks. Morris water maze experiment was used to detect the cognitive function of rats. Tm-Vision behavioral experiment system was used to observe the behavioral changes of rats,and the hippocampal neuronal line structure was observed by transmission electron microscope. Mitochondrial complex colorimetry was adopted to detect rats C Ⅰ,C Ⅱ,C Ⅲ,C Ⅳ activity. Western blot was used to detect the expression of mitochondrial adenosine monophosphate-activated protein kinase( AMPK) protein in the hippocampus. Result: Compared with normal group,model group rats latent escape period time increasing,through fewer,movement distance,movement time increasing,center residence time increased( P < 0. 05,P <0. 01),mitochondria C Ⅱ,C Ⅲ,C Ⅳ activity decline( P < 0. 05,P < 0. 01),The AMPK protein expression increased significantly( P < 0. 05,P < 0. 01). Compared with model group,Sijunzi Tang medium and high dose group and dihydroergot decoction group showed decreased latency,increased crossing times,decreased exercise distance,decreased exercise time,and decreased center residence time( P < 0. 05,P < 0. 01). Compared with model group C Ⅱ,mitochondrial complex CⅣ activity increases,AMPK protein expression lowered( P < 0. 05,P < 0. 01),Sijunzi Tang high dose group of mitochondrial complex C Ⅱ,C Ⅲ activity increased significantly,AMPK protein expression significantly lowered( P < 0. 05,P < 0. 01). Conclusion: The abnormal behavior of AD rats may be related to the decrease of central hippocampal energy metabolism and mitochondrial function. Sijunzi Tang has therapeutic effect.
引文
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[11]秦莉霞,吴倩,夏鹏,等.参芪益智颗粒对Aβ1-42双侧海马注射阿尔茨海默病模型大鼠PI3K/AKT信号通路的影响[J].世界科学技术-中医药现代化,2018,20(12):2161-2166.
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[13]陶一鸣,杜艳军,田青,等.电针对阿尔茨海默病大鼠齿状回星形胶质细胞和白细胞介素-10表达及其共定位的影响[J].中国老年学杂志,2019,39(4):874-878.
[14]刘静,杜艳军,周清莲,等.针加灸对阿尔茨海默病大鼠海马区蛋白酪氨酸激酶-2/信号转导和转录激活因子-3信号通路的影响[J].针刺研究,2019,44(2):79-84.
[15] Pilon G,Dallaire P,Marette A. Inhibition of inducible nitric-oxide synthase by activators of AMP-activated protein kinase:a new mechanism of action of insulinsensitizing drugs[J]. J Biol Chem,2004,279(20):20767-20774.
[16]王懿,张艳,礼海.益气活血方干预PGC-1α调控心衰心肌细胞能量代谢重构的作用机制[J].中国实验方剂学杂志,2015,21(6):169-173.
[17] Ueno H, Nakazato M. The mechanistic relationship between the vagal afferent pathway,central nervous system and peripheral organs in appetite regulation[J].J Diabetes Investig,2016,7(6):812-818.
[2] Majd S, Jht P. Oxidative stress and decreased mitochondrial superoxide dismutase 2 and peroxiredoxin1 and 4 based mechanism of concurrent activation of AMPK and mTOR in Alzheimer's disease[J]. Curr Alzheimer Res,2018,19(10):764-776.
[3]刘文俊,陈伟仁,宋囡,等.脾气虚模型大鼠海马与下丘脑神经元线粒体分裂因子和线粒体分裂蛋白1的表达[J].中国中西医结合杂志,2017,37(11):1356-1360.
[4]刘旭东.四君子汤介导c AMP-AMPK通路对脾气虚证大鼠海马呼吸链酶活性影响研究[D].沈阳:辽宁中医药大学,2015.
[5]黄继汉,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[J].中国临床药理学与治疗学,2004,9(9):1069-1072.
[6]刘旭东,刘文俊,孙大宇,等.脾气虚证模型大鼠神疲乏力的客观化评价[J].中华中医药杂志,2015,30(3):699-701.
[7]刘明,刘杨,邓颖,等.蓝布正提取物对血管性痴呆大鼠学习记忆能力及海马NT-3,BDNF蛋白表达的影响[J].中国实验方剂学杂志,2017,23(17):154-158.
[8] Kopeikina K J,Carlson G A,Pitstick R,et al[J]. Tau accumulation causes mitochondrial distribution deficits in neurons in a mouse model of tauopathy and in Human Alzheimer's disease brain[J]. Am J Pathol,2011,179(4):2071-2082
[9] Ferreira S T,Lourenco M V,Oliveira M M,et al.Soluble amyloid-beta oligomers as synaptotoxins leading to cognitive impairment in Alzheimer's disease[J].Front Cell Neurosci,2015,9(16):191.
[10] Waqar A. Overlapped metabolic and therapeutic links between Alzheimer and diabetes[J]. Mol Neurobiol,2013,47(1):399-424.
[11]秦莉霞,吴倩,夏鹏,等.参芪益智颗粒对Aβ1-42双侧海马注射阿尔茨海默病模型大鼠PI3K/AKT信号通路的影响[J].世界科学技术-中医药现代化,2018,20(12):2161-2166.
[12]刘明,刘杨,邓颖,等.蓝布正提取物对血管性痴呆大鼠学习记忆能力及海马NT-3,BDNF蛋白表达的影响[J].中国实验方剂学杂志,2017,23(17):154-158.
[13]陶一鸣,杜艳军,田青,等.电针对阿尔茨海默病大鼠齿状回星形胶质细胞和白细胞介素-10表达及其共定位的影响[J].中国老年学杂志,2019,39(4):874-878.
[14]刘静,杜艳军,周清莲,等.针加灸对阿尔茨海默病大鼠海马区蛋白酪氨酸激酶-2/信号转导和转录激活因子-3信号通路的影响[J].针刺研究,2019,44(2):79-84.
[15] Pilon G,Dallaire P,Marette A. Inhibition of inducible nitric-oxide synthase by activators of AMP-activated protein kinase:a new mechanism of action of insulinsensitizing drugs[J]. J Biol Chem,2004,279(20):20767-20774.
[16]王懿,张艳,礼海.益气活血方干预PGC-1α调控心衰心肌细胞能量代谢重构的作用机制[J].中国实验方剂学杂志,2015,21(6):169-173.
[17] Ueno H, Nakazato M. The mechanistic relationship between the vagal afferent pathway,central nervous system and peripheral organs in appetite regulation[J].J Diabetes Investig,2016,7(6):812-818.
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