黑果枸杞和白刺的抗焦虑作用及机制探讨
发布时间：2018-08-22 08:06
【摘要】：目的焦虑症(Anxiety disorder)又称之为焦虑性神经症(Anxiety neurosis),是一种以焦虑情绪为主的神经疾病,以持续性的焦虑、紧张、惊恐不安等焦虑情绪为表现,并且伴有自主神经紊乱、肌肉紧张以及运动不安等症状。焦虑症在临床上主要分为广泛性焦虑症(Generalized anxiety disorder,GAD)、惊恐障碍(Panic disorder,PD)、社交恐惧症(Social anxiety disorder,SAD)、强迫症(Obsessive compulsive disorder,OCD)、创伤后应激障碍(Post-traumatic stress disorder,PTSD)和广场恐惧症(Agoraphobia)等。焦虑症发病率逐年增加,已成为现代疾病谱的常见病和多发病,严重影响人们的生活质量,因此抗焦虑药物及作用机理研究逐渐成为学术界研究热点。黑果枸杞来源于茄科枸杞属植物黑果枸杞(Lycium ruthenicum Murray)的成熟果实,在藏医药经典著作《四部医典》、《晶珠本草》中均有记载,性平、味甘,功效清心热,主治心脏病、妇科病、月经不调等。白刺来源于蒺藜科白刺属植物白刺(Nitraria tangutorum Bobr.)的成熟果实,其地域分布、外形、口感、功效与黑果枸杞相似,而藏药中两者也可混用替代,均称之为"旁玛"。黑果枸杞和白刺均富含花色苷类天然成分,现代药理学研究认为,花色苷作为一种类黄酮物质,具有显著的生理活性如清除自由基和抗氧化、预防心血管疾病、预防神经退行性疾病和改善认知提高记忆力等。富含花色苷类成分的黑果枸杞和白刺是否具有抗焦虑的潜力,抗焦虑药效如何?主要的抗焦虑作用机制是什么?这些问题很值得我们研究。方法1.制备黑果枸杞提取物(LRE)和白刺提取物(NTE),采用大孔树脂吸附法富集和除杂,采用pH示差法对LRE和NTE中的总花色苷含量作定量分析。2.通过小鼠高架十字迷宫实验(EPM)、小鼠明暗箱实验(LDB)、空瓶应激(EBS)致焦虑大鼠行为学实验以及束缚应激(RS)焦虑大鼠行为学实验,探究和比较黑果枸杞提取物(LRE)和白刺提取物(NTE)抗焦虑药效。3.基于空瓶应激致焦虑大鼠模型探讨比较LRE和NTE对大鼠血清下丘脑-垂体-肾上腺轴(HPA)的影响。4.基于束缚应激致焦虑大鼠模型探讨比较LRE和NTE对大鼠皮层和海马脑内源性营养因子(BDNF)蛋白表达和胞外调节蛋白激酶(ERK)通路的影响,对海马凋亡相关因子表达的影响,对海马神经元再生增殖的影响。结果主要研究内容及结果如下:1.LRE和NTE的制备1.1 LRE和NTE的提取参考文献方法,实验确定LRE和NTE的提取条件为:25倍含2%甲酸的80%乙醇溶液50℃避光浸提两次,每次3 h。1.2 LRE和NTE的纯化 AB-8大孔树脂吸附法除杂和富集,回收溶剂冷冻干燥,得率分别为8.04%和5.02%。1.3 LRE和NTE的含量测定采用pH示差法对LRE和NTE中的总花色苷含量作了定量分析,结果表明,以矢车菊素-3-O-葡萄糖苷计,LRE和NTE中的总花色苷含量分别为 439.07±4.61 mg/100g 和 222.13±1.70 mg/100 g(以生药量计,干重)。2.药效学研究2.1 LRE和NTE对小鼠抗焦虑作用研究小鼠高架十字迷宫实验结果显示,与空白对照组相比,LRE的剂量为320 mg/kg/d、NTE的剂量为400 mg/kg/d时,可显著增加进入开臂时间和次数百分比。明暗箱实验结果显示,与空白对照组相比,LRE的剂量为320mg/kg/d、NTE的剂量为400mg/kg/d时,可显著增加小鼠在明箱中的时间与进入明箱中的次数。2.2 LRE和NTE对空瓶应激致焦虑大鼠抗焦虑作用研究实验结果表明,与空白对照组相比,模型组可显著降低大鼠进入高架十字迷宫开臂时间百分比和开臂次数百分比以及大鼠进入旷场中央区的次数和在中央区停留的时间,说明造模成功。空瓶应激后高架十字迷宫实验结果表明,同模型组相比,LRE低、高剂量组和NTE低、高剂量组进入开臂次数均显著升高;LRE低、高剂量组和NTE高剂量组进入开臂时间均显著升高。空瓶应激后旷场实验结果表明,同模型组相比,LRE低、高剂量组和NTE低、高剂量组进入中央区次数均显著升高;LRE高剂量组、NTE高剂量组进入中央区时间均显著升高。2.3 LRE和NTE对束缚应激致焦虑大鼠抗焦虑作用研究采用束缚应激对大鼠进行焦虑造模,应用高架十字迷宫实验(EPM)进一步评价和比较LRE和NTE的抗焦虑作用。实验结果表明,与空白组相比,模型组显著降低大鼠开臂次数和时间百分比,说明造模成功,大鼠出现焦虑情绪。与模型组相比,LRE低、高剂量组显著增加大鼠进入开臂次数和时间百分比。与模型组相比,NTE低剂量组可显著增加大鼠进入开臂次数百分比;NTE高剂量组可显著增加大鼠进入开臂次数和进入开臂时间百分比。3.机制探讨3.1 LRE和NTE对空瓶应激模型大鼠下丘脑-垂体-肾上腺轴的影响。采用酶联免疫吸附法检测了 LRE和NTE对空瓶应激大鼠血清HPA轴相关激素促肾上腺皮质激素释放因子(CRF)、肾上腺皮质激素(ACTH)、皮质酮(CORT)含量,结果显示,大鼠空瓶应激模型能引起HPA轴亢进,表现为CRF、ACTH、CORT含量相较于空白组显著增加。与模型组相比,LRE低剂量组和高剂量组以及NTE高剂量组能显著降低空瓶应激大鼠血清HPA轴各激素含量。据此推测LRE和NTE抗焦虑作用的机制可能是:抑制应激所造成的HPA轴各激素含量升高,缓解HPA轴的过度亢奋,从而达到抗焦虑作用效果。3.2 LRE和NTE对束缚应激模型大鼠脑内ERK/MAPK通路的影响。通过蛋白印迹法检测LRE和NTE对束缚应激致焦虑模型大鼠脑内ERK/MAPK通路的影响,即胞外信号调节蛋白激酶(ERK)和环磷酸腺苷反应元件结合蛋白(CREB)磷酸化水平变化。结果表明,与空白组相比,模型组大鼠皮层和海马中的P-ERK与p-CREB水平显著升高;与模型组相比,LRE低剂量组和高剂量组可显著减少大鼠皮层和海马中p-ERK与p-CREB的表达水平,NTE高剂量组可显著减少大鼠皮层和海马中p-ERK与p-CREB的表达水平。这一结果提示LRE和NTE可能通过调节ERK/MAPK通路发挥其抗焦虑作用。3.3 LRE和NTE对束缚应激模型大鼠脑内BDNF蛋白表达的影响。通过蛋白印迹测定法检测了 LRE和NTE对束缚应激致焦虑模型大鼠海马和皮层BDNF蛋白表达的影响,结果显示,与空白组相比,模型组的BDNF表达水平显著下降,与模型组相比,LRE低剂量组和高剂量组以及NTE高剂量组能使大鼠的BDNF表达水平显著上升,这表明,BDNF参与了束缚应激致焦虑信号,而LRE和NTE可通过调节BDNF产生抗焦虑作用。此外,文献报道,ERK/MAPK通路与BDNF蛋白的表达关系密切,ERK/MAPK通路的变化可以影响BDNF蛋白含量的表达,而实验结果表明LRE和NTE对ERK/MAPK通路和BDNF蛋白的表达都有显著的影响,因此提示LRE和NTE也可能通过调节它们的相互作用而发挥其抗焦虑药效。3.4 LRE和NTE对束缚应激模型大鼠海马神经元凋亡相关因子的影响。通过蛋白印迹法检测了 LRE和NTE对束缚应激致焦虑模型大鼠海马凋亡相关因子Bax和Bcl-2表达的影响。实验结果显示,与空白组相比,模型组凋亡抑制因子Bcl-2显著降低,而促凋亡因子Bax表达显著增加,提示束缚应激造模可能诱导大鼠海马神经元细胞的凋亡。与模型组相比,LRE高剂量组和NTE高剂量组均能显著降低大鼠海马中促凋亡因子Bax的蛋白表达水平,LRE低剂量组和高剂量组以及NTE低剂量组和高剂量组均能显著增加大鼠海马中凋亡抑制因子Bcl-2的蛋白表达水平。有研究认为,大鼠海马BDNF蛋白表达与凋亡相关因子的表达有较强的相关性,结合实验结果,提示束缚应激造模可能会影响动物海马BDNF蛋白和凋亡相关因子的表达水平,而花色苷类成分LRE和NTE可能通过影响动物神经元的凋亡和BDNF蛋白的表达而起到抗焦虑的神经生物学效应。3.5 LRE和NTE对束缚应激模型大鼠海马神经元再生增殖的影响。在束缚应激后高架十字迷宫实验结束后,各组大鼠腹腔注射5-溴-2'-脱氧尿苷(BrdU)标记海马S期先祖细胞,以反映齿状回(DG区)细胞的增殖情况,断头取脑,制作石蜡切片,进行免疫组织化学处理,对各组大鼠海马齿状回BrdU阳性细胞进行计数。实验结果显示,与空白组相比,模型组大鼠海马齿状回BrdU阳性细胞计数显著降低,提示束缚应激造模减少了大鼠海马齿状回细胞增殖。与模型组相比,LRE低剂量组和高剂量组以及NTE高剂量组能显著增加海马齿状回BrdU阳性细胞计数,提示预给药LRE和NTE可减缓大鼠因束缚应激造成的海马齿状回细胞增殖减缓,从而达到促进海马神经元细胞再生增殖的效果。结论本课题分别采用两个经典小鼠行为学实验(EPM、LDB)探讨比较LRE和NTE的抗焦虑药效,并通过空瓶应激致焦虑模型和束缚应激致焦虑模型的大鼠行为学实验对LRE和NTE的抗焦虑作用进行了进一步的验证。结果表明,LRE和NTE在以上各个不同模型上均表现出稳定的抗焦虑作用;同剂量条件下,LRE较NTE表现出更稳定的抗焦虑药效。在机制探讨方面,通过探讨比较LRE和NTE对空瓶应激后大鼠HPA轴的影响,推测它们可能通过抑制模型动物HPA系统功能亢进来减轻动物焦虑状态;通过探讨比较LRE和NTE对束缚应激后大鼠脑内BDNF/ERK/MAPK通路的影响,对海马凋亡相关因子的影响,对海马神经元再生增殖的影响,推测LRE和NTE可能通过抑制ERK和CREB的过度磷酸化,促进BDNF蛋白的表达,调节海马凋亡相关因子表达,促进海马神经元再生增殖,进而提高海马神经元可塑性来达到抗焦虑的作用。通过以上研究,明确LRE和NTE抗焦虑作用和探讨相关的作用机制,为它们有效应用于抗焦虑药物的研发提供实验依据。
[Abstract]:Objective Anxiety disorder, also known as Anxiety neurosis, is a neurological disorder characterized by anxiety. It is characterized by persistent anxiety, tension, panic and other anxiety emotions, accompanied by autonomic nervous disorder, muscle tension and motor disturbance. Generalized anxiety disorder (GAD), panic disorder (PD), social anxiety disorder (SAD), obsessive compulsive disorder (OCD), post-traumatic stress disorder (PTSD) and agoraphobia are the main causes of anxiety disorder. It has become a common disease and frequently-occurring disease in modern disease spectrum, seriously affecting people's quality of life, so the study of anti-anxiety drugs and their mechanism of action has gradually become a focus of academic research. It is recorded in Pearl Herb > that it has plain sex, sweet taste, heart-clearing effect, cardiac disease, gynecological disease and irregular menstruation. White thorn originates from the mature fruit of Nitraria tangutorum Bobr. Its geographical distribution, shape, taste and efficacy are similar to those of black fruit Lycium barbarum. Chinese wolfberry and white thorn are rich in natural anthocyanins. Modern pharmacological studies show that anthocyanins, as a flavonoid, have significant physiological activities, such as scavenging free radicals and antioxidant, preventing cardiovascular diseases, preventing neurodegenerative diseases and improving cognition and memory. Whether Lycium barbarum and White thorn have the potential of anti-anxiety, and what are the main anti-anxiety mechanisms? These problems are worth studying. Methods 1. LRE and NTE were prepared, enriched and impurities removed by macroporous resin adsorption, and total anthocyanins in LRE and NTE were determined by pH difference method. Quantitative analysis was conducted. 2. Anti-anxiety effects of LRE and NTE were investigated and compared by EPM, LDB, EBS and RS in mice. To compare the effects of LRE and NTE on serum hypothalamic-pituitary-adrenal axis (HPA) in rats. 4. To compare the effects of LRE and NTE on the expression of endogenous nutrient factor (BDNF) protein and extracellular regulated protein kinase (ERK) pathway in rat cortex and hippocampus and the expression of apoptosis-related factors in hippocampus based on restraint stress-induced anxiety rat model. Results The main contents and results were as follows: 1. Preparation of LRE and NTE 1.1 LRE and extraction of NTE reference method, the extraction conditions of LRE and NTE were determined as follows: 25 times of 2% formic acid 80% ethanol solution was extracted twice at 50, each time 3 h.1.2 LRE and NTE purified AB-8 macroporous resin. The yield of 1.3 LRE and NTE was 8.04% and 5.02% respectively. The content of total anthocyanins in LRE and NTE was determined by pH differential method. The results showed that the content of total anthocyanins in LRE and NTE were 439.07 (+4.61 mg/100g) and 222.6 mg/100g respectively by cyanidin-3-O-glucoside. Pharmacodynamic study 2.1 LRE and NTE on anti-anxiety effect in mice The results of elevated maze test in mice showed that the dose of LRE was 320 mg/kg/d and NTE was 400 mg/kg/d, which could significantly increase the time and frequency of entering the arm. The results showed that compared with the blank control group, LRE dosage was 320 mg/kg/d, NTE dosage was 400 mg/kg/d, the mice in the open box time and the number of times into the open box significantly increased. 2.2 LRE and NTE on empty bottle stress-induced anxiety rats anti-anxiety effect experimental results showed that compared with the blank control group, the model group can significantly reduce the rats'anxiety. The percentage of arm opening time, percentage of arm opening times, the number of rats entering the center of the open field and the time spent in the center of the open field indicated that the model was successful. After empty bottle stress, the open field test showed that LRE was lower, high dose group and NTE was lower, and the number of times the high dose group entered the central area was significantly higher than that of the model group. The antianxiety effects of LRE and NTE were evaluated and compared by EPM. The results showed that the number of open arms and the percentage of time in the model group were significantly lower than those in the blank group. Compared with the model group, the LRE was lower, and the high dose group increased the number of times and percentage of time to open arms. Compared with the model group, the NTE low dose group significantly increased the percentage of times to open arms; the NTE high dose group significantly increased the number of times to open arms and percentage of time to open arms. The effects of 3.1 LRE and NTE on hypothalamus-pituitary-adrenal axis in empty bottle stress rats were studied. The effects of LRE and NTE on serum levels of HPA axis-related hormones, corticotrophin-releasing factor (CRF), adrenocorticotrophin (ACTH) and corticosterone (CORT) in empty bottle stress rats were detected by enzyme-linked immunosorbent assay (ELISA). Compared with the model group, LRE low-dose group, high-dose group and NTE high-dose group could significantly reduce the levels of hormones in HPA axis in serum of empty bottle stress rats. The effects of LRE and NTE on the ERK/MAPK pathway in the brain of restraint stress rats were studied by Western blot. The effects of LRE and NTE on the ERK/MAPK pathway in the brain of restraint stress-induced anxiety rats were detected by Western blot. The results showed that the levels of P-ERK and p-CREB in the cortex and hippocampus of rats in the model group were significantly higher than those in the blank group, and the levels of p-ERK and p-CREB in the cortex and hippocampus of rats in the low-dose group and the high-dose group were significantly lower than those in the model group. The results suggest that LRE and NTE may play an anti-anxiety role by regulating the ERK/MAPK pathway. 3.3 LRE and NTE affect the expression of BDNF protein in the brain of restraint stress model rats. The effects of LRE and NTE on the expression of BDNF protein in the brain of restraint stress model rats were detected by Western blot. Considering the effect of BDNF protein expression in hippocampus and cortex of model rats, the results showed that the expression of BDNF in model group was significantly lower than that in blank group. Compared with model group, the expression of BDNF in low-dose LRE group, high-dose LRE group and high-dose NTE group increased significantly, which indicated that BDNF participated in restraint stress-induced anxiety signal and LR was involved in restraint stress-induced anxiety signal. In addition, it has been reported that ERK/MAPK pathway is closely related to the expression of BDNF protein. The changes of ERK/MAPK pathway can affect the expression of BDNF protein. The experimental results show that both LRE and NTE have significant effects on the expression of ERK/MAPK pathway and BDNF protein. The effects of LRE and NTE on the expression of apoptosis-related factors Bax and Bcl-2 in hippocampus of restraint stress-induced anxiety model rats were studied by Western blotting. Compared with the model group, the expression of Bcl-2 and Bax in the hippocampus of rats were significantly decreased in the model group, while the expression of Bax was significantly increased in the model group, suggesting that restraint stress modeling may induce apoptosis of rat hippocampal neurons. The expression of Bcl-2 protein in hippocampus of rats was significantly increased in both high-dose and low-dose NTE groups. It was suggested that the expression of BDNF protein in hippocampus of rats was strongly correlated with the expression of apoptosis-related factors. The expression levels of BDNF protein and apoptosis-related factors in hippocampus, and anthocyanins LRE and NTE may play an anti-anxiety neurobiological effect by affecting the apoptosis of neurons and the expression of BDNF protein. 3.5 LRE and NTE affect the regeneration and proliferation of hippocampal neurons in restraint stress model rats. After the labyrinth experiment, rats in each group were intraperitoneally injected with 5-bromo-2'-deoxyuridine (BrdU) to label S-phase progenitor cells of the hippocampus to reflect the proliferation of cells in the dentate gyrus (DG area). The brain was cut off, paraffin sections were made, and the BrdU positive cells in the dentate gyrus of the hippocampus were counted by immunohistochemical method. Compared with the model group, the BrdU positive cells in dentate gyrus of hippocampus were significantly decreased in the model group, suggesting that restraint stress modeling reduced the proliferation of dentate gyrus cells in the hippocampus. The proliferation of hippocampal dentate gyrus cells was slowed down due to restraint stress in rats, and the effect of promoting the regeneration and proliferation of hippocampal neurons was achieved. Conclusion The antianxiety effects of LRE and NTE were compared by two classical behavioral experiments in mice (EPM, LDB). The anxious model induced by empty bottle stress and the anxious model induced by restraint stress were established. The results showed that LRE and NTE showed stable anti-anxiety effects in the above different models; LRE showed more stable anti-anxiety effects than NTE at the same dose. In terms of mechanism, LRE and NTE were compared to empty bottles. The effects of LRE and NTE on BDNF/ERK/MAPK pathway, apoptosis-related factors in hippocampus and the regeneration and proliferation of hippocampal neurons were investigated and compared. E may inhibit the hyperphosphorylation of ERK and CREB, promote the expression of BDNF protein, regulate the expression of apoptosis-related factors in hippocampus, promote the regeneration and proliferation of hippocampal neurons, and then improve the plasticity of hippocampal neurons to achieve the anti-anxiety effect. It can provide experimental evidence for the development of anxiolytic drugs.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5
本文编号：2196506
[Abstract]:Objective Anxiety disorder, also known as Anxiety neurosis, is a neurological disorder characterized by anxiety. It is characterized by persistent anxiety, tension, panic and other anxiety emotions, accompanied by autonomic nervous disorder, muscle tension and motor disturbance. Generalized anxiety disorder (GAD), panic disorder (PD), social anxiety disorder (SAD), obsessive compulsive disorder (OCD), post-traumatic stress disorder (PTSD) and agoraphobia are the main causes of anxiety disorder. It has become a common disease and frequently-occurring disease in modern disease spectrum, seriously affecting people's quality of life, so the study of anti-anxiety drugs and their mechanism of action has gradually become a focus of academic research. It is recorded in Pearl Herb > that it has plain sex, sweet taste, heart-clearing effect, cardiac disease, gynecological disease and irregular menstruation. White thorn originates from the mature fruit of Nitraria tangutorum Bobr. Its geographical distribution, shape, taste and efficacy are similar to those of black fruit Lycium barbarum. Chinese wolfberry and white thorn are rich in natural anthocyanins. Modern pharmacological studies show that anthocyanins, as a flavonoid, have significant physiological activities, such as scavenging free radicals and antioxidant, preventing cardiovascular diseases, preventing neurodegenerative diseases and improving cognition and memory. Whether Lycium barbarum and White thorn have the potential of anti-anxiety, and what are the main anti-anxiety mechanisms? These problems are worth studying. Methods 1. LRE and NTE were prepared, enriched and impurities removed by macroporous resin adsorption, and total anthocyanins in LRE and NTE were determined by pH difference method. Quantitative analysis was conducted. 2. Anti-anxiety effects of LRE and NTE were investigated and compared by EPM, LDB, EBS and RS in mice. To compare the effects of LRE and NTE on serum hypothalamic-pituitary-adrenal axis (HPA) in rats. 4. To compare the effects of LRE and NTE on the expression of endogenous nutrient factor (BDNF) protein and extracellular regulated protein kinase (ERK) pathway in rat cortex and hippocampus and the expression of apoptosis-related factors in hippocampus based on restraint stress-induced anxiety rat model. Results The main contents and results were as follows: 1. Preparation of LRE and NTE 1.1 LRE and extraction of NTE reference method, the extraction conditions of LRE and NTE were determined as follows: 25 times of 2% formic acid 80% ethanol solution was extracted twice at 50, each time 3 h.1.2 LRE and NTE purified AB-8 macroporous resin. The yield of 1.3 LRE and NTE was 8.04% and 5.02% respectively. The content of total anthocyanins in LRE and NTE was determined by pH differential method. The results showed that the content of total anthocyanins in LRE and NTE were 439.07 (+4.61 mg/100g) and 222.6 mg/100g respectively by cyanidin-3-O-glucoside. Pharmacodynamic study 2.1 LRE and NTE on anti-anxiety effect in mice The results of elevated maze test in mice showed that the dose of LRE was 320 mg/kg/d and NTE was 400 mg/kg/d, which could significantly increase the time and frequency of entering the arm. The results showed that compared with the blank control group, LRE dosage was 320 mg/kg/d, NTE dosage was 400 mg/kg/d, the mice in the open box time and the number of times into the open box significantly increased. 2.2 LRE and NTE on empty bottle stress-induced anxiety rats anti-anxiety effect experimental results showed that compared with the blank control group, the model group can significantly reduce the rats'anxiety. The percentage of arm opening time, percentage of arm opening times, the number of rats entering the center of the open field and the time spent in the center of the open field indicated that the model was successful. After empty bottle stress, the open field test showed that LRE was lower, high dose group and NTE was lower, and the number of times the high dose group entered the central area was significantly higher than that of the model group. The antianxiety effects of LRE and NTE were evaluated and compared by EPM. The results showed that the number of open arms and the percentage of time in the model group were significantly lower than those in the blank group. Compared with the model group, the LRE was lower, and the high dose group increased the number of times and percentage of time to open arms. Compared with the model group, the NTE low dose group significantly increased the percentage of times to open arms; the NTE high dose group significantly increased the number of times to open arms and percentage of time to open arms. The effects of 3.1 LRE and NTE on hypothalamus-pituitary-adrenal axis in empty bottle stress rats were studied. The effects of LRE and NTE on serum levels of HPA axis-related hormones, corticotrophin-releasing factor (CRF), adrenocorticotrophin (ACTH) and corticosterone (CORT) in empty bottle stress rats were detected by enzyme-linked immunosorbent assay (ELISA). Compared with the model group, LRE low-dose group, high-dose group and NTE high-dose group could significantly reduce the levels of hormones in HPA axis in serum of empty bottle stress rats. The effects of LRE and NTE on the ERK/MAPK pathway in the brain of restraint stress rats were studied by Western blot. The effects of LRE and NTE on the ERK/MAPK pathway in the brain of restraint stress-induced anxiety rats were detected by Western blot. The results showed that the levels of P-ERK and p-CREB in the cortex and hippocampus of rats in the model group were significantly higher than those in the blank group, and the levels of p-ERK and p-CREB in the cortex and hippocampus of rats in the low-dose group and the high-dose group were significantly lower than those in the model group. The results suggest that LRE and NTE may play an anti-anxiety role by regulating the ERK/MAPK pathway. 3.3 LRE and NTE affect the expression of BDNF protein in the brain of restraint stress model rats. The effects of LRE and NTE on the expression of BDNF protein in the brain of restraint stress model rats were detected by Western blot. Considering the effect of BDNF protein expression in hippocampus and cortex of model rats, the results showed that the expression of BDNF in model group was significantly lower than that in blank group. Compared with model group, the expression of BDNF in low-dose LRE group, high-dose LRE group and high-dose NTE group increased significantly, which indicated that BDNF participated in restraint stress-induced anxiety signal and LR was involved in restraint stress-induced anxiety signal. In addition, it has been reported that ERK/MAPK pathway is closely related to the expression of BDNF protein. The changes of ERK/MAPK pathway can affect the expression of BDNF protein. The experimental results show that both LRE and NTE have significant effects on the expression of ERK/MAPK pathway and BDNF protein. The effects of LRE and NTE on the expression of apoptosis-related factors Bax and Bcl-2 in hippocampus of restraint stress-induced anxiety model rats were studied by Western blotting. Compared with the model group, the expression of Bcl-2 and Bax in the hippocampus of rats were significantly decreased in the model group, while the expression of Bax was significantly increased in the model group, suggesting that restraint stress modeling may induce apoptosis of rat hippocampal neurons. The expression of Bcl-2 protein in hippocampus of rats was significantly increased in both high-dose and low-dose NTE groups. It was suggested that the expression of BDNF protein in hippocampus of rats was strongly correlated with the expression of apoptosis-related factors. The expression levels of BDNF protein and apoptosis-related factors in hippocampus, and anthocyanins LRE and NTE may play an anti-anxiety neurobiological effect by affecting the apoptosis of neurons and the expression of BDNF protein. 3.5 LRE and NTE affect the regeneration and proliferation of hippocampal neurons in restraint stress model rats. After the labyrinth experiment, rats in each group were intraperitoneally injected with 5-bromo-2'-deoxyuridine (BrdU) to label S-phase progenitor cells of the hippocampus to reflect the proliferation of cells in the dentate gyrus (DG area). The brain was cut off, paraffin sections were made, and the BrdU positive cells in the dentate gyrus of the hippocampus were counted by immunohistochemical method. Compared with the model group, the BrdU positive cells in dentate gyrus of hippocampus were significantly decreased in the model group, suggesting that restraint stress modeling reduced the proliferation of dentate gyrus cells in the hippocampus. The proliferation of hippocampal dentate gyrus cells was slowed down due to restraint stress in rats, and the effect of promoting the regeneration and proliferation of hippocampal neurons was achieved. Conclusion The antianxiety effects of LRE and NTE were compared by two classical behavioral experiments in mice (EPM, LDB). The anxious model induced by empty bottle stress and the anxious model induced by restraint stress were established. The results showed that LRE and NTE showed stable anti-anxiety effects in the above different models; LRE showed more stable anti-anxiety effects than NTE at the same dose. In terms of mechanism, LRE and NTE were compared to empty bottles. The effects of LRE and NTE on BDNF/ERK/MAPK pathway, apoptosis-related factors in hippocampus and the regeneration and proliferation of hippocampal neurons were investigated and compared. E may inhibit the hyperphosphorylation of ERK and CREB, promote the expression of BDNF protein, regulate the expression of apoptosis-related factors in hippocampus, promote the regeneration and proliferation of hippocampal neurons, and then improve the plasticity of hippocampal neurons to achieve the anti-anxiety effect. It can provide experimental evidence for the development of anxiolytic drugs.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5
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