【摘要】：目的焦慮癥(Anxiety disorder)又稱之為焦慮性神經(jīng)癥(Anxiety neurosis),是一種以焦慮情緒為主的神經(jīng)疾病,以持續(xù)性的焦慮、緊張、驚恐不安等焦慮情緒為表現(xiàn),并且伴有自主神經(jīng)紊亂、肌肉緊張以及運(yùn)動不安等癥狀。焦慮癥在臨床上主要分為廣泛性焦慮癥(Generalized anxiety disorder,GAD)、驚恐障礙(Panic disorder,PD)、社交恐懼癥(Social anxiety disorder,SAD)、強(qiáng)迫癥(Obsessive compulsive disorder,OCD)、創(chuàng)傷后應(yīng)激障礙(Post-traumatic stress disorder,PTSD)和廣場恐懼癥(Agoraphobia)等。焦慮癥發(fā)病率逐年增加,已成為現(xiàn)代疾病譜的常見病和多發(fā)病,嚴(yán)重影響人們的生活質(zhì)量,因此抗焦慮藥物及作用機(jī)理研究逐漸成為學(xué)術(shù)界研究熱點。黑果枸杞來源于茄科枸杞屬植物黑果枸杞(Lycium ruthenicum Murray)的成熟果實,在藏醫(yī)藥經(jīng)典著作《四部醫(yī)典》、《晶珠本草》中均有記載,性平、味甘,功效清心熱,主治心臟病、婦科病、月經(jīng)不調(diào)等。白刺來源于蒺藜科白刺屬植物白刺(Nitraria tangutorum Bobr.)的成熟果實,其地域分布、外形、口感、功效與黑果枸杞相似,而藏藥中兩者也可混用替代,均稱之為"旁瑪"。黑果枸杞和白刺均富含花色苷類天然成分,現(xiàn)代藥理學(xué)研究認(rèn)為,花色苷作為一種類黃酮物質(zhì),具有顯著的生理活性如清除自由基和抗氧化、預(yù)防心血管疾病、預(yù)防神經(jīng)退行性疾病和改善認(rèn)知提高記憶力等。富含花色苷類成分的黑果枸杞和白刺是否具有抗焦慮的潛力,抗焦慮藥效如何?主要的抗焦慮作用機(jī)制是什么?這些問題很值得我們研究。方法1.制備黑果枸杞提取物(LRE)和白刺提取物(NTE),采用大孔樹脂吸附法富集和除雜,采用pH示差法對LRE和NTE中的總花色苷含量作定量分析。2.通過小鼠高架十字迷宮實驗(EPM)、小鼠明暗箱實驗(LDB)、空瓶應(yīng)激(EBS)致焦慮大鼠行為學(xué)實驗以及束縛應(yīng)激(RS)焦慮大鼠行為學(xué)實驗,探究和比較黑果枸杞提取物(LRE)和白刺提取物(NTE)抗焦慮藥效。3.基于空瓶應(yīng)激致焦慮大鼠模型探討比較LRE和NTE對大鼠血清下丘腦-垂體-腎上腺軸(HPA)的影響。4.基于束縛應(yīng)激致焦慮大鼠模型探討比較LRE和NTE對大鼠皮層和海馬腦內(nèi)源性營養(yǎng)因子(BDNF)蛋白表達(dá)和胞外調(diào)節(jié)蛋白激酶(ERK)通路的影響,對海馬凋亡相關(guān)因子表達(dá)的影響,對海馬神經(jīng)元再生增殖的影響。結(jié)果主要研究內(nèi)容及結(jié)果如下:1.LRE和NTE的制備1.1 LRE和NTE的提取參考文獻(xiàn)方法,實驗確定LRE和NTE的提取條件為:25倍含2%甲酸的80%乙醇溶液50℃避光浸提兩次,每次3 h。1.2 LRE和NTE的純化 AB-8大孔樹脂吸附法除雜和富集,回收溶劑冷凍干燥,得率分別為8.04%和5.02%。1.3 LRE和NTE的含量測定采用pH示差法對LRE和NTE中的總花色苷含量作了定量分析,結(jié)果表明,以矢車菊素-3-O-葡萄糖苷計,LRE和NTE中的總花色苷含量分別為 439.07±4.61 mg/100g 和 222.13±1.70 mg/100 g(以生藥量計,干重)。2.藥效學(xué)研究2.1 LRE和NTE對小鼠抗焦慮作用研究小鼠高架十字迷宮實驗結(jié)果顯示,與空白對照組相比,LRE的劑量為320 mg/kg/d、NTE的劑量為400 mg/kg/d時,可顯著增加進(jìn)入開臂時間和次數(shù)百分比。明暗箱實驗結(jié)果顯示,與空白對照組相比,LRE的劑量為320mg/kg/d、NTE的劑量為400mg/kg/d時,可顯著增加小鼠在明箱中的時間與進(jìn)入明箱中的次數(shù)。2.2 LRE和NTE對空瓶應(yīng)激致焦慮大鼠抗焦慮作用研究實驗結(jié)果表明,與空白對照組相比,模型組可顯著降低大鼠進(jìn)入高架十字迷宮開臂時間百分比和開臂次數(shù)百分比以及大鼠進(jìn)入曠場中央?yún)^(qū)的次數(shù)和在中央?yún)^(qū)停留的時間,說明造模成功�？掌繎�(yīng)激后高架十字迷宮實驗結(jié)果表明,同模型組相比,LRE低、高劑量組和NTE低、高劑量組進(jìn)入開臂次數(shù)均顯著升高;LRE低、高劑量組和NTE高劑量組進(jìn)入開臂時間均顯著升高。空瓶應(yīng)激后曠場實驗結(jié)果表明,同模型組相比,LRE低、高劑量組和NTE低、高劑量組進(jìn)入中央?yún)^(qū)次數(shù)均顯著升高;LRE高劑量組、NTE高劑量組進(jìn)入中央?yún)^(qū)時間均顯著升高。2.3 LRE和NTE對束縛應(yīng)激致焦慮大鼠抗焦慮作用研究采用束縛應(yīng)激對大鼠進(jìn)行焦慮造模,應(yīng)用高架十字迷宮實驗(EPM)進(jìn)一步評價和比較LRE和NTE的抗焦慮作用。實驗結(jié)果表明,與空白組相比,模型組顯著降低大鼠開臂次數(shù)和時間百分比,說明造模成功,大鼠出現(xiàn)焦慮情緒。與模型組相比,LRE低、高劑量組顯著增加大鼠進(jìn)入開臂次數(shù)和時間百分比。與模型組相比,NTE低劑量組可顯著增加大鼠進(jìn)入開臂次數(shù)百分比;NTE高劑量組可顯著增加大鼠進(jìn)入開臂次數(shù)和進(jìn)入開臂時間百分比。3.機(jī)制探討3.1 LRE和NTE對空瓶應(yīng)激模型大鼠下丘腦-垂體-腎上腺軸的影響。采用酶聯(lián)免疫吸附法檢測了 LRE和NTE對空瓶應(yīng)激大鼠血清HPA軸相關(guān)激素促腎上腺皮質(zhì)激素釋放因子(CRF)、腎上腺皮質(zhì)激素(ACTH)、皮質(zhì)酮(CORT)含量,結(jié)果顯示,大鼠空瓶應(yīng)激模型能引起HPA軸亢進(jìn),表現(xiàn)為CRF、ACTH、CORT含量相較于空白組顯著增加。與模型組相比,LRE低劑量組和高劑量組以及NTE高劑量組能顯著降低空瓶應(yīng)激大鼠血清HPA軸各激素含量。據(jù)此推測LRE和NTE抗焦慮作用的機(jī)制可能是:抑制應(yīng)激所造成的HPA軸各激素含量升高,緩解HPA軸的過度亢奮,從而達(dá)到抗焦慮作用效果。3.2 LRE和NTE對束縛應(yīng)激模型大鼠腦內(nèi)ERK/MAPK通路的影響。通過蛋白印跡法檢測LRE和NTE對束縛應(yīng)激致焦慮模型大鼠腦內(nèi)ERK/MAPK通路的影響,即胞外信號調(diào)節(jié)蛋白激酶(ERK)和環(huán)磷酸腺苷反應(yīng)元件結(jié)合蛋白(CREB)磷酸化水平變化。結(jié)果表明,與空白組相比,模型組大鼠皮層和海馬中的P-ERK與p-CREB水平顯著升高;與模型組相比,LRE低劑量組和高劑量組可顯著減少大鼠皮層和海馬中p-ERK與p-CREB的表達(dá)水平,NTE高劑量組可顯著減少大鼠皮層和海馬中p-ERK與p-CREB的表達(dá)水平。這一結(jié)果提示LRE和NTE可能通過調(diào)節(jié)ERK/MAPK通路發(fā)揮其抗焦慮作用。3.3 LRE和NTE對束縛應(yīng)激模型大鼠腦內(nèi)BDNF蛋白表達(dá)的影響。通過蛋白印跡測定法檢測了 LRE和NTE對束縛應(yīng)激致焦慮模型大鼠海馬和皮層BDNF蛋白表達(dá)的影響,結(jié)果顯示,與空白組相比,模型組的BDNF表達(dá)水平顯著下降,與模型組相比,LRE低劑量組和高劑量組以及NTE高劑量組能使大鼠的BDNF表達(dá)水平顯著上升,這表明,BDNF參與了束縛應(yīng)激致焦慮信號,而LRE和NTE可通過調(diào)節(jié)BDNF產(chǎn)生抗焦慮作用。此外,文獻(xiàn)報道,ERK/MAPK通路與BDNF蛋白的表達(dá)關(guān)系密切,ERK/MAPK通路的變化可以影響B(tài)DNF蛋白含量的表達(dá),而實驗結(jié)果表明LRE和NTE對ERK/MAPK通路和BDNF蛋白的表達(dá)都有顯著的影響,因此提示LRE和NTE也可能通過調(diào)節(jié)它們的相互作用而發(fā)揮其抗焦慮藥效。3.4 LRE和NTE對束縛應(yīng)激模型大鼠海馬神經(jīng)元凋亡相關(guān)因子的影響。通過蛋白印跡法檢測了 LRE和NTE對束縛應(yīng)激致焦慮模型大鼠海馬凋亡相關(guān)因子Bax和Bcl-2表達(dá)的影響。實驗結(jié)果顯示,與空白組相比,模型組凋亡抑制因子Bcl-2顯著降低,而促凋亡因子Bax表達(dá)顯著增加,提示束縛應(yīng)激造�？赡苷T導(dǎo)大鼠海馬神經(jīng)元細(xì)胞的凋亡。與模型組相比,LRE高劑量組和NTE高劑量組均能顯著降低大鼠海馬中促凋亡因子Bax的蛋白表達(dá)水平,LRE低劑量組和高劑量組以及NTE低劑量組和高劑量組均能顯著增加大鼠海馬中凋亡抑制因子Bcl-2的蛋白表達(dá)水平。有研究認(rèn)為,大鼠海馬BDNF蛋白表達(dá)與凋亡相關(guān)因子的表達(dá)有較強(qiáng)的相關(guān)性,結(jié)合實驗結(jié)果,提示束縛應(yīng)激造�？赡軙绊憚游锖ｑRBDNF蛋白和凋亡相關(guān)因子的表達(dá)水平,而花色苷類成分LRE和NTE可能通過影響動物神經(jīng)元的凋亡和BDNF蛋白的表達(dá)而起到抗焦慮的神經(jīng)生物學(xué)效應(yīng)。3.5 LRE和NTE對束縛應(yīng)激模型大鼠海馬神經(jīng)元再生增殖的影響。在束縛應(yīng)激后高架十字迷宮實驗結(jié)束后,各組大鼠腹腔注射5-溴-2'-脫氧尿苷(BrdU)標(biāo)記海馬S期先祖細(xì)胞,以反映齒狀回(DG區(qū))細(xì)胞的增殖情況,斷頭取腦,制作石蠟切片,進(jìn)行免疫組織化學(xué)處理,對各組大鼠海馬齒狀回BrdU陽性細(xì)胞進(jìn)行計數(shù)。實驗結(jié)果顯示,與空白組相比,模型組大鼠海馬齒狀回BrdU陽性細(xì)胞計數(shù)顯著降低,提示束縛應(yīng)激造模減少了大鼠海馬齒狀回細(xì)胞增殖。與模型組相比,LRE低劑量組和高劑量組以及NTE高劑量組能顯著增加海馬齒狀回BrdU陽性細(xì)胞計數(shù),提示預(yù)給藥LRE和NTE可減緩大鼠因束縛應(yīng)激造成的海馬齒狀回細(xì)胞增殖減緩,從而達(dá)到促進(jìn)海馬神經(jīng)元細(xì)胞再生增殖的效果。結(jié)論本課題分別采用兩個經(jīng)典小鼠行為學(xué)實驗(EPM、LDB)探討比較LRE和NTE的抗焦慮藥效,并通過空瓶應(yīng)激致焦慮模型和束縛應(yīng)激致焦慮模型的大鼠行為學(xué)實驗對LRE和NTE的抗焦慮作用進(jìn)行了進(jìn)一步的驗證。結(jié)果表明,LRE和NTE在以上各個不同模型上均表現(xiàn)出穩(wěn)定的抗焦慮作用;同劑量條件下,LRE較NTE表現(xiàn)出更穩(wěn)定的抗焦慮藥效。在機(jī)制探討方面,通過探討比較LRE和NTE對空瓶應(yīng)激后大鼠HPA軸的影響,推測它們可能通過抑制模型動物HPA系統(tǒng)功能亢進(jìn)來減輕動物焦慮狀態(tài);通過探討比較LRE和NTE對束縛應(yīng)激后大鼠腦內(nèi)BDNF/ERK/MAPK通路的影響,對海馬凋亡相關(guān)因子的影響,對海馬神經(jīng)元再生增殖的影響,推測LRE和NTE可能通過抑制ERK和CREB的過度磷酸化,促進(jìn)BDNF蛋白的表達(dá),調(diào)節(jié)海馬凋亡相關(guān)因子表達(dá),促進(jìn)海馬神經(jīng)元再生增殖,進(jìn)而提高海馬神經(jīng)元可塑性來達(dá)到抗焦慮的作用。通過以上研究,明確LRE和NTE抗焦慮作用和探討相關(guān)的作用機(jī)制,為它們有效應(yīng)用于抗焦慮藥物的研發(fā)提供實驗依據(jù)。
[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.
【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285.5

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