基于海马糖原代谢和星形胶质细胞结构可塑性研究人参总皂苷抗应激致抑郁样行为的机理

发布时间：2018-05-12 06:12

本文选题：星形胶质细胞 + 可塑性　；参考：《南京中医药大学》2017年硕士论文

【摘要】：研究目的观察人参总皂苷(GTS)对皮质酮(CORT)诱导的小鼠抑郁样行为以及海马糖原代谢和星型胶质细胞(AS)结构可塑性损伤的影响,探讨GTS抗抑郁的作用机理。研究方法实验1.基于体视学技术研究抑郁模型小鼠海马星形胶质细胞的结构可塑性20只C57BL/6N小鼠随机分成2组,即对照组(Control)、模型组(CORT),每组10只。CORT组连续5周皮下注射C0RT制备小鼠抑郁模型,CORT组随机时间点给予皮下注射CORT,注射剂量为20 mg · kg-1 · d-1。Control组给予相同剂量的生理盐水。造模成功后,进行行为学试验、血清CORT含量测定。将每组小鼠进行心脏灌注后取脑,用于胶质纤维酸性蛋白(GFAP)免疫组化染色和体视学方法定量海马体积、GFAP阳性细胞的数目、体积和细胞突起长度。实验2.抑郁模型小鼠海马组织中糖原水平的测定30只C57BL/6N小鼠随机分成2组,即对照组(Control)、模型组(CORT),每组15只。造模成功后,将每组动物随机分成三小组,每组5只,第一小组进行脑糖原(GC)含量和糖原磷酸化酶-脑型(PYGM)、糖原合成酶(GS)活性的检测。第二小组小鼠处死方法与第一组相同,取海马组织装入EP管中,采用Western-blot法检测糖原磷酸化酶-脑型(PYGM)和糖原合成酶(GS)蛋白的表达。第三小组处死方法与第一组相同,取海马组织装入EP管中,采用Q-PCR法检测糖原磷酸化酶-脑型(PYGM)和糖原合成酶(GS)基因水平的表达。实验3.GTS对抑郁模型小鼠星形胶质细胞结构可塑性损伤的机制研究90只C57BL/6N 小鼠随机分成 6 组:Control、CORT、CORT+氟西汀 FLU(10mg·kg·kg-1·d-1)、CORT+低剂量组 GTSL(12.5 mg · kg-1 · d-1)、CORT+中剂量组 GTSM(25 mg · kg-1 · d-1)以及CORT+高剂量组GTSH(50 mg · kg-1 · d-1),每组15只。FLU或GTS各剂量组给予灌胃,持续21天。造模成功后在第20天和第21天分别进行强迫游泳实验(Forced Swimming Test,FST)和强迫悬尾实验(Suspenseful Test,TST),第22天对每只小鼠进行眼眶取血,待测血清CORT含量水平,第23天将所有动物按照实验要求处死。每组动物随机分成三小组,每组5只,第一小组心脏灌注后取脑,用于GFAP免疫组化染色和体视学方法定量海马体积、GFAP阳性细胞的数目、体积和细胞突起长度。第二小组将小鼠投入液氮中处死,取出后迅速剥离大脑,在液氮中把海马组织研磨成粉后,取海马组织装入EP管中,进行脑糖原(GC)含量和糖原磷酸化酶-脑型(PYGM)、糖原合成酶(GS)活性的检测。第三小组小鼠处死方法与第二组相同,取海马组织装入EP管中,用于检测PYGM和GS蛋白和基因的表达。实验4.GTS对正常小鼠星形胶质细胞结构可塑性损伤的机制研究20只C57BL/6N小鼠随机分成2组:Control、GTSH组,每组10只。高剂量组GTSH(50 mg · kg-1 · d-1)给予灌胃,持续21天。造模成功后在第22天将所有动物按照实验要求处死。每组动物随机分成二小组,每组5只,第一小组心脏灌注后取脑,用于GFAP免疫组化染色和体视学方法定量海马体积、GFAP阳性细胞的数目、体积和细胞突起长度。第二小组将小鼠投入液氮中处死,取出后迅速剥离大脑,在液氮中把海马组织研磨成粉后,取海马组织装入EP管中,进行糖原含量的检测。研究结果实验1.长期皮质酮注射诱导小鼠抑郁样行为,损伤海马星形胶质细胞结构可塑性与Control组相比,CORT组小鼠强迫游泳和强迫悬尾的静止时间明显延长(P0.01),血清CORT含量显著升高(P0.01)。小鼠海马体视学检测结果进行t检验:①与Control组相比,CORT组小鼠海马体积明著缩小(P0.01),GFAP阳性AS的数目显著减少(P0.01),GFAP阳性AS的突起长度明显缩短(P0.01)。②免疫组化GFAP染色与Control组相比,CORT组小鼠海马GFAP阳性AS的体积明显缩小(P0.01)。实验2.长期皮质酮注射下调海马组织中糖原的水平与Control组相比,CORT组小鼠海马GC含量显著降低(P0.01),海马PYGM活性显著升高(P0.01),海马GS活性显著升高(P0.01)。与Control组相比,CORT组小鼠海马GS蛋白含量表达显著升高(P0.01),海马PYGM蛋白含量表达显著升高(P0.01),海马GS基因含量表达显著升高(P0.01),海马PYGM基因含量表达显著升高(P0.01)。实验3.GTS可上调抑郁模型小鼠海马组织中糖原的水平,拮抗星形胶质细胞结构可塑性的损伤CORT组血清皮质酮水平高于Control组(P0.01),造模期间给予各剂量GTS对血清皮质酮水平均无显著影响。FLU、GTS 3个剂量组均能明显拮抗模型小鼠的FST和TST(均P0.01),以及缩短静止不动时间,并显著性抑制模型小鼠海马PYGM的活性(P0.05,P0.01、P0.05、P0.05)。与CORT组相比,FLU、GTSL、GTSH组均能显著上调模型小鼠海马组织GC含量(均P0.01)。与CORT组相比,FLU、GTSM、GTSH组均能显著上调模型小鼠海马组织GS的活性(均P0.01),显著拮抗模型小鼠海马组织内PYGM蛋白的表达,均P0.01),且显著下调模型小鼠海马组织内PYGM基因的表达(均P0.01)。与CORT组相比,FLU、GTSM、GTSH组均能显著上调模型小鼠海马组织内GS蛋白的表达,且显著上调模型小鼠海马组织内GS基因的表达(P0.05,p＜0.05,P0.01)。与CORT组比,FLU、GTSM、GTSH组的模型小鼠海马体积明显增大(均P0.01),海马GFAP阳性细胞数目明显增加(均P0.01)。与CORT组比,FLU、GTSH组的模型小鼠海马GFAP阳性AS的体积增大(均P0.05),海马GFAP阳性AS的突起长度显著增加(均P0.01)。实验4.GTS不影响正常小鼠海马星形胶质细胞的结构可塑性每组每只小鼠海马体视学检测结果进行t检验:①与Control组相比,海马GC含量水平无明显变化(P0.05)。②与Control组相比,GTSH组小鼠海马体积无明显变化(P0.05),海马GFAP阳性AS的数目无明显变化(P0.05),海马GFAP阳性AS的体积无明显变化(P0.05),海马GFAP阳性AS的突起长度无明显变化(P0.05),研究结论本实验采用长期CORT注射模拟慢性应激,复制抑郁动物模型,观察GTS抗抑郁的作用,并从海马糖原代谢和星形胶质细胞结构可塑性角度探讨机制。研究结果显示长期C0RT能诱导抑郁样行为并损伤海马星形胶质细胞结构可塑性。GTS抗抑郁作用有如下特点:①具有抗抑郁样行为作用,如通过行为学FST和TST实验,高剂量GTS能减少皮质酮抑郁模型小鼠的抑郁样行为。②通过增加AS内糖原含量拮抗海马损伤。③上调皮质酮抑郁模型小鼠海马组织内GS蛋白和基因表达、下调皮质酮抑郁模型小鼠海马组织内PYGM蛋白和基因表达,表明高剂量GTS可使糖原在蛋白和基因水平上同时增加。④提高小鼠海马体积、GFAP阳性细胞数目、GFAP阳性细胞的细胞体积、GFAP阳性细胞的突起长度,改善海马AS可塑性。本研究认为,GTS抗抑郁的作用可能与改善AS的糖原含量和结构可塑性有关。
[Abstract]:Objective To observe the effect of Panax ginseng total saponins (GTS) on the depressive behavior of mice induced by corticosterone (CORT), the effect of hippocampal glycogen metabolism and the structural plasticity of astrocytes (AS), and to explore the mechanism of GTS antidepressant action. Method 1. study method experiment based on stereology to study the structure of astrocytes in the hippocampus of depressive model mice 20 C57BL/6N mice were randomly divided into 2 groups, that is, the control group (Control), the model group (CORT), each group of 10.CORT groups was injected with C0RT for 5 weeks to prepare the mice depression model, and the CORT group was given the subcutaneous injection of CORT at random time point, and the injection amount was 20 mg. Kg-1 d-1.Control group given the same dose of saline. Behavior test and determination of serum CORT content. The brain was taken after heart perfusion in each group. The volume of hippocampus, the number of GFAP positive cells, volume and the length of cell protuberance were quantified by immunohistochemical staining and stereological method of glial fibrillary acidic protein (GFAP). The level of glycogen in hippocampus of experimental 2. depressed model mice was measured by 30 C5 7BL/6N mice were randomly divided into 2 groups, namely, the control group (Control), the model group (CORT), 15 in each group. After the success of the model, each group was randomly divided into three groups, each group was 5. The first group was tested for the content of brain glycogen (GC), glycogen phosphorylase brain (PYGM), and glycogen synthase (GS) activity. The second group mice were killed in the same way as the first group. The expression of glycogen phosphorylase brain type (PYGM) and glycogen synthetase (GS) protein was detected by Western-blot method. The third group was killed in the same way as the first group. The hippocampal tissue was loaded into the EP tube, and the expression of glycogen phosphorylase brain (PYGM) and glycogen synthetase (GS) gene was detected by Q-PCR. 90 C57BL/6N mice were randomly divided into 6 groups: Control, CORT, CORT+ fluoxetine FLU (10mg. Kg-1 D-1), CORT+ low dose group GTSL (12.5), and high dose group (50). Kg-1. D-1), each group of 15.FLU or GTS dose groups were given the gastric lavage for 21 days. After the success of the model, the forced swimming test (Forced Swimming Test, FST) and the forced tail suspension experiment (Suspenseful Test, TST) were performed respectively, and the blood of each mouse was taken into the orbit for twenty-second days, and the level of the serum CORT content was measured, and the twenty-third days would be done. Animals were killed in accordance with the experimental requirements. Each group of animals was randomly divided into three groups, each group was 5. After the first group of heart perfusion, the brain was taken. GFAP immunohistochemical staining and stereological methods were used to quantify the volume of the hippocampus, the number of GFAP positive cells, the volume and the length of the cell protuberance. The second group killed the mice in liquid nitrogen and quickly stripped off. After grinding the hippocampus into powder in the liquid nitrogen, the hippocampus was loaded into the EP tube to detect the content of GC and the activity of glycogen phosphorylase brain (PYGM) and glycogen synthase (GS). The third group of mice were executed in the same way as the second groups, and the hippocampus group was woven into the EP tube to detect the expression of PYGM and GS proteins and genes. Study the mechanism of plastic damage of astrocyte structure in normal mice by experimental 4.GTS, 20 C57BL/6N mice were randomly divided into 2 groups: Control, group GTSH, 10 in each group. GTSH (50 mg. Kg-1. D-1) was given to the stomach for 21 days in the high dose group. All the animals were killed at twenty-second days after the model was successful. The animals of each group were randomly divided into two groups. The two group, each group of 5, the first group after heart perfusion to take the brain, used for GFAP immunohistochemical staining and stereological methods to quantify the volume of hippocampus, the number of GFAP positive cells, volume and cell protuberance length. Group second killed the mice in liquid nitrogen, removed the brain quickly, and ground the hippocampus into powder in liquid nitrogen and took the hippocampus. The tissue was loaded into EP tube to detect the content of glycogen. Results 1. a long-term corticosterone injection induced depressive behavior in mice. Compared with the Control group, the plasticity of hippocampal astrocytes was significantly longer than that of the Control group (P0.01), and the serum CORT content was significantly increased (P0.01). The results of t test in rat hippocampus: (1) compared with group Control, the volume of hippocampus in CORT group was reduced (P0.01), the number of GFAP positive AS decreased significantly (P0.01), and the length of GFAP positive AS significantly shortened (P0.01). 0.01) in experiment 2., compared with group Control, the level of glycogen in hippocampus of long-term corticosterone injection was significantly decreased (P0.01), PYGM activity in hippocampus was significantly increased (P0.01) and hippocampal GS activity increased significantly (P0.01). Compared with group Control, the expression of GS protein in hippocampus of CORT mice increased significantly (P0.01), hippocampus The expression of protein content increased significantly (P0.01), the expression of GS gene content in hippocampus was significantly increased (P0.01), and the expression of PYGM gene in hippocampus increased significantly (P0.01). Experimental 3.GTS could increase the level of glycogen in the hippocampus of depression model mice, and antagonize the plasticity of astrocytes in the CORT group, the serum corticosterone level was higher than that of the Control group (P0.01). Every dose of GTS had no significant effect on the level of serum corticosterone during the process of modeling.FLU, and the 3 doses of GTS could obviously antagonize the FST and TST of the model mice (all P0.01), and shorten the resting time, and significantly inhibit the activity of PYGM in the hippocampus of the model mice (P0.05, P0.01, P0.05, P0.05). The content of GC in hippocampus of model mice was increased (P0.01). Compared with group CORT, FLU, GTSM and GTSH could significantly increase the activity of GS in the hippocampus of model mice (P0.01), significantly antagonize the expression of PYGM protein in the hippocampus of model mice, all P0.01), and significantly down the expression of PYGM gene in the hippocampus of model mice. Compared with the group FLU, GTSM and GTSH, the expression of GS protein in the hippocampus of the model mice was significantly up-regulated, and the expression of GS gene in the hippocampus of the model mice was significantly increased (P0.05, P < 0.05, P0.01). Compared with the CORT group, FLU, GTSM, the hippocampal volume of the model mice of the GTSH group increased significantly (all of them). 1). Compared with group CORT, the volume of GFAP positive AS in hippocampus of model mice of FLU and GTSH increased (P0.05), and the protuberance length of hippocampal GFAP positive AS increased significantly (all P0.01). Experimental 4.GTS did not affect the structural plasticity of astrocytes in the hippocampus of normal mice. There was no significant change in the level of hippocampal GC content (P0.05). Compared with the Control group, there was no significant change in the volume of hippocampus in the GTSH group (P0.05), the number of GFAP positive AS in the hippocampus was not significantly changed (P0.05), and the volume of the GFAP positive AS in the hippocampus was not significantly changed (P0.05), and there was no obvious change in the length of the hippocampal positive AS. The conclusion of the study was adopted in this experiment. Long term CORT injection simulated chronic stress, replicated depressive animal model, observed the antidepressant effect of GTS, and explored the mechanism of hippocampal glycogen metabolism and astrocyte structural plasticity. The results showed that long-term C0RT could induce depressive behavior and damage the antidepressant effect of hippocampus astrocytic structure plastic.GTS as follows. Points: (1) it has antidepressant behavior, such as through the behavior FST and TST experiments, high dose GTS can reduce the depressive behavior of the corticosterone depression model mice. (2) antagonize the hippocampus injury by increasing the content of glycogen in AS. (3) up regulation of GS protein and gene expression in the hippocampus of corticosterone depression model mice, and down-regulation of corticosterone depression model mice The expression of PYGM protein and gene in the hippocampus indicates that high dose of GTS can increase glycogen at the level of protein and gene. 4. Increase the volume of the hippocampus, the number of GFAP positive cells, the cell volume of the GFAP positive cells, the protuberance length of the GFAP positive cells, and improve the plasticity of AS in the hippocampus. This study suggests that the antidepressant effect of GTS may be modified by the modification of GTS. The glycogen content of good AS is related to the plasticity of the structure.

【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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