丙戊酸鈉聯(lián)合雌激素對去卵巢癡呆小鼠的作用及其機制的初步研究
發(fā)布時間：2018-08-27 09:57
【摘要】：目的:研究雌激素缺乏不同時間段癡呆模型小鼠腦內(nèi)雌激素受體(Estrogen receptors,ERs)及糖原合成酶激酶3β(glycogen synthase kinase 3β)的動態(tài)變化,并觀察丙戊酸鈉(valproic acid sodium salt,VPA)聯(lián)合雌激素對APP/PS1雙轉(zhuǎn)基因去勢AD模型小鼠腦內(nèi)雌激素受體含量及GSK-3β相關(guān)信號通路的影響,探討相關(guān)的作用機制。本課題研究成果將為進一步揭示AD的發(fā)病機制及臨床上研究和開發(fā)治療AD的多靶向藥物提供新的實驗室依據(jù)。方法:本課題分兩部分進行。第一部分:選取3月齡雌性APP/PS1雙轉(zhuǎn)基因小鼠40只,將其隨機分為假手術(shù)組(AD-Sham組)和手術(shù)組(AD-OVX組),同月齡野生型小鼠為對照組(WT組),每組10只。分別于術(shù)后1個月和術(shù)后5個月,采用多種方法檢測小鼠外周血雌激素水平、行為學、腦組織形態(tài)學及相關(guān)蛋白的變化。第二部分:選取去勢后1個月癡呆模型小鼠進行藥物治療,隨機平均分為6組:生理鹽水對照組、VPA藥物處理組、E2藥物處理組、Liq藥物處理組、VPA聯(lián)合E2藥物處理組及VPA聯(lián)合Liq藥物處理組,每組8只。vpa藥物處理組小鼠給予藥物vpa30mg/kg·d,雌激素藥物處理組給予e2藥物2.4ug/只·d,liq治療組給予liq50ug/kg·d,vpa和e2聯(lián)合治療組給予vpa30mg/kg·d和e22.4ug/只·d,vpa和liq聯(lián)合治療組給予vpa30mg/kg·d和liq50ug/kg·d,生理鹽水治療組給予等量生理鹽水,除liq給藥方式為灌胃外,其余給藥均為腹腔注射。藥物處理1個月后,采用高架十字迷宮及曠場實驗以檢測各組小鼠的抑郁焦慮行為;morris水迷宮檢測小鼠的空間學習及記憶能力;利用蛋白印跡(westernblot)等技術(shù),檢測聯(lián)合用藥對小鼠腦內(nèi)雌激素受體含量,gsk-3β活性及老年斑的影響,并探討其相關(guān)機制。結(jié)果:1.第一部分實驗結(jié)果:ovx1個月和5個月組ad小鼠體循環(huán)雌激素水平顯著下調(diào)(p0.05);ovx1個月組小鼠子宮無明顯縮小,而ovx5個月出現(xiàn)明顯萎縮。morris水迷宮顯示,ovx1個月后,與ad-sham組相比,ad-ovx組小鼠找到平臺的逃避潛伏期和路程顯著增加(p0.05),跨越平臺的次數(shù)明顯減小(p0.05)。隨著ovx時間的延長,至ovx5個月后,ad-ovx組小鼠找到平臺的時間和經(jīng)過的路程更進一步延長(p0.05),跨越平臺的次數(shù)也相應下降(p0.05)。免疫熒光結(jié)果顯示,與ad-sham組相比,ad-ovx1個月和5個月組小鼠腦內(nèi)老年斑均顯著增加(p0.05)。westernblot結(jié)果顯示,與ad-sham組相比,ad-ovx1個月及5個月組小鼠腦內(nèi)erβ蛋白水平出現(xiàn)進行性下調(diào)(p0.05);相應地,ad-ovx組小鼠腦內(nèi)p-gsk-3β水平降低的趨勢與erβ一致(p0.05),盡管正常小鼠和ad小鼠腦內(nèi)gsk-3β總蛋白無明顯差異(p0.05)。2.第二部分實驗結(jié)果:1、行為學1)高架十字迷宮檢測結(jié)果顯示:聯(lián)合藥物處理組小鼠在開放臂活動次數(shù)明顯增多(p0.05),聯(lián)合藥物處理組在開放臂停留的時間與其余各組相比均明顯延長(p0.01)。在開放臂活動次數(shù)及開放臂停留時間的比率上,vpa與e2聯(lián)合處理組均明顯增加(p0.01),liq處理組也明顯增加(p0.05);vpa與e2,vpa與liq聯(lián)合處理組較vpa及e2單獨藥物處理組也顯著增加(p0.05)。2)水迷宮實驗結(jié)果:可視平臺條件下,各組藥物處理小鼠找到平臺的時間和路程均無明顯的差異(p0.05)。隱蔽平臺實驗條件下,其他各藥物處理組小鼠找到隱蔽平臺的游動時間及路程與生理鹽水對照組相比均明顯縮短(p0.05);而聯(lián)合藥物處理組游動時間及距離更加縮短(p0.05)�？臻g探索實驗中,與生理鹽水組比較,聯(lián)合藥物處理組穿越平臺的次數(shù)明顯增多(p0.01),單一藥物處理組也明顯增多(p0.05);與聯(lián)合藥物處理組比較,vpa處理組和liq處理組均明顯減少(p0.05),e2處理組顯著減少(p0.01)。2、各組小鼠子宮形態(tài)及重量統(tǒng)計結(jié)果顯示:各藥物處理組小鼠子宮在外觀形態(tài)上均有不同程度的增大,統(tǒng)計分析結(jié)果得出,vpa組,liq組與control組相比,子宮重量無明顯變化(p0.05);e2組,vpa+e2組,vpa+liq與control組相比,子宮重量顯著增加(p0.01)。e2組,vpa+e2組小鼠子宮形態(tài)與其余各組相比明顯增生,重量也顯著增加(p0.05),并有大小形狀不一的結(jié)節(jié)形成。3.蛋白水平實驗結(jié)果:(1)雌激素受體α與β與生理鹽水組比較,大腦皮質(zhì)區(qū)erα蛋白含量在聯(lián)合藥物處理組中顯著上升(p0.01),在各單獨藥物處理組中無明顯差異(p0.05)。與生理鹽水對照組相比,大腦海馬區(qū)erα蛋白水平在vpa藥物處理組中無明顯變化(p0.05),在其余各藥物處理組中均顯著增多(p0.05)。在erβ含量的比較中,與生理鹽水對照組相比,其余各藥物處理組大腦皮質(zhì)及海馬區(qū)內(nèi)erβ含量均顯著增多(p0.05),且聯(lián)合藥物處理組大腦皮質(zhì)及海馬區(qū)內(nèi)erβ的含量比各藥物單獨處理組明顯增多(p0.05)。(2)gsk-3β活性檢測:與生理鹽水對照組相比,其余各單獨藥物處理組小鼠腦皮質(zhì)及海馬區(qū)gsk-3β總蛋白水平均無明顯差異(p0.05),聯(lián)合藥物處理組腦皮質(zhì)區(qū)gsk-3β總蛋白水平顯著下降(p0.05),而腦海馬區(qū)內(nèi)gsk-3β總蛋白水平無明顯差異(p0.05)。其第9位點磷酸化水平標志著gsk-3β活性的高低,與生理鹽水對照組相比,vpa組、e2組腦皮質(zhì)區(qū)pser9-gsk-3β水平均無明顯差異(p0.05),liq組及聯(lián)合藥物處理組腦皮質(zhì)區(qū)pser9-gsk-3β水平明顯升高(p0.05);而在各組腦海馬區(qū)pser9-gsk-3β水平的比較中,與生理鹽水對照組相比,其余各藥物處理組pser9-gsk-3β水平均明顯升高(p0.05),且與各單獨藥物處理組相比,聯(lián)合藥物處理組pser9-gsk-3β水平也顯著升高(p0.05)。結(jié)論:1、卵巢切除手術(shù)后,ad小鼠體循環(huán)雌激素水平隨年齡增長呈下降趨勢,子宮形態(tài)隨年齡增長逐漸萎縮。2、卵巢切除手術(shù)后,AD小鼠空間學習記憶能力隨年齡增長逐漸下降。3、卵巢切除手術(shù)后,AD小鼠腦內(nèi)Aβ的沉積及老年斑的形成增多,其可能機制可能是與ERβ含量下降所導致的GSK-3β活性異常有關(guān)。4、藥物處理明顯改善了去勢AD小鼠的抑郁焦慮行為,緩解了其空間學習記憶能力的下降,且聯(lián)合用藥的效果明顯優(yōu)于單一藥物處理。5、藥物處理顯著增加了去勢AD小鼠腦皮質(zhì)及海馬區(qū)雌激素受體的含量,且聯(lián)合用藥的效果更顯著。6、藥物處理顯著增加了去勢AD小鼠腦皮質(zhì)及海馬區(qū)p ser9-GSK-3β的含量,使GSK-3β的活性受到了抑制,且聯(lián)合用藥的效果更顯著。
[Abstract]:AIM: To study the dynamic changes of estrogen receptors (ERs) and glycogen synthase kinase 3 beta (GSK 3 beta) in the brain of mice with estrogen deficiency at different time points, and to observe the effect of valproic acid sodium salt (VPA) combined with estrogen on the estrogen in the brain of APP / PS1 castrated AD model mice. The results of this study will provide a new laboratory basis for further revealing the pathogenesis of AD and clinical research and development of multi-targeted drugs for AD. METHODS: This study was carried out in two parts. Part I: Selection of 3-month-old female APP/PS1. Forty double transgenic mice were randomly divided into sham operation group (AD-Sham group) and operation group (AD-OVX group) and wild type mice of the same age group (WT group) with 10 mice in each group. Part: One month after ovariectomy, dementia model mice were randomly divided into 6 groups: normal saline control group, VPA drug treatment group, E2 drug treatment group, Liq drug treatment group, VPA combined with E2 drug treatment group and VPA combined with Liq drug treatment group, with 8 mice in each group. The treatment group was given E2 drug 2.4ug/kg/d, the liq treatment group was given liq50ug/kg/d, the VPA and E2 combined treatment group were given VPA 30mg/kg/d and e22.4ug/kg/d, the VPA and liq combined treatment group were given VPA 30mg/kg/d and liq50ug/kg/d, and the saline treatment group was given the same amount of saline, except the liq was given intraperitoneally. After one month of treatment, the depression and anxiety behaviors of mice in each group were tested by elevated cross maze and open field test; the spatial learning and memory abilities of mice were tested by Morris water maze; the estrogen receptor content, GSK-3 beta activity and senile plaque were detected by Western blot and other techniques. Results: 1. the results of the first part of the experiment: OVX 1 month and 5 months group AD mice circulatory estrogen levels significantly decreased (p0.05); OVX 1 month group mice uterus did not significantly shrink, but OVX 5 months showed significant atrophy. Morris water maze showed that OVX 1 month later, compared with ad-sham group, ad-ovx mice found a platform escape potential. The incubation period and distance increased significantly (p0.05), and the number of crossing platforms decreased significantly (p0.05). with the prolongation of OVX time, to OVX 5 months later, the time of finding platforms and the distance of crossing platforms in ad-ovx group were further prolonged (p0.05), and the number of crossing platforms decreased accordingly (p0.05). immunofluorescence results showed that compared with ad-sham group, ad-ovx 1. The results of Western blot showed that compared with ad-sham group, the level of ER-beta protein in the brain of ad-ovx mice decreased progressively (p0.05); accordingly, the decrease of p-gsk-3 beta level in the brain of ad-ovx mice was consistent with that of normal mice and AD mice (p0.05). There was no significant difference in the total protein of GSK-3 beta between the two groups (p0.05). 2. Part 2: 1, Behavior 1) The results of the elevated maze test showed that the number of activities in the open arm of the mice in the combined drug treatment group was significantly increased (p0.05), and the duration of stay in the open arm of the combined drug treatment group was significantly longer than that of the other groups (p0.01). The ratio of activity times and residence time of open arms in the VPA and E2 combined treatment group were significantly increased (p0.01), and the liq treatment group was also significantly increased (p0.05); VPA and e2, VPA and liq combined treatment group compared with the VPA and E2 single drug treatment group were significantly increased (p0.05). There was no significant difference in the time and distance between the two groups (p0.05). under the covert platform experiment, the swimming time and distance of the mice in the other drug treatment groups were significantly shorter than those in the normal saline control group (p0.05), while the swimming time and distance of the combined drug treatment group were shorter (p0.05). Compared with the saline group, the number of crossing platform in the combined drug treatment group was significantly increased (p0.01), and the number of crossing platform in the single drug treatment group was also significantly increased (p0.05); compared with the combined drug treatment group, VPA treatment group and liq treatment group were significantly reduced (p0.05), E2 treatment group was significantly reduced (p0.01). uterine morphology and weight statistics of mice in each group showed that: The uterus morphology of the mice in the treatment group increased in different degrees. the results of statistical analysis showed that the weight of uterus in VPA group, liq group and control group had no significant change (p0.05); the weight of uterus in E2 group, VPA + E2 group, VPA + liq group and control group increased significantly (p0.01). The results of protein level experiment: (1) compared with the normal saline and estrogen receptor alpha group, the content of ER alpha protein in the cerebral cortex increased significantly in the combined drug treatment group (p0.01), and there was no significant difference in the single drug treatment group (p0.05). Compared with the control group, the level of ER-alpha protein in the cerebral hippocampus did not change significantly in the VPA treatment group (p0.05), but increased significantly in the other drug treatment groups (p0.05). in the comparison of ER-beta content, compared with the normal saline control group, the content of ER-beta in the cerebral cortex and hippocampus of the other drug treatment groups increased significantly (p0.05). (2) GSK-3 beta activity test: compared with the normal saline control group, there was no significant difference in the levels of GSK-3 beta total protein in the cerebral cortex and hippocampus of mice treated with other drugs alone (p0.05), and there was no significant difference in GSK-3 beta total protein between the combined drug treatment group and the normal saline control group (p0.05). The total protein level decreased significantly (p0.05), but there was no significant difference in the total protein level of GSK-3 beta in the hippocampus (p0.05). the phosphorylation level of the ninth locus marked the activity of GSK-3 beta. compared with the normal saline control group, the levels of pser9-gsk-3 beta in the VPA group, E2 group and liq group had no significant difference (p0.05), and the combined drug treatment group. The levels of pser9-gsk-3 beta in the hippocampus of each group were significantly higher than those in the normal saline group (p0.05). The levels of pser9-gsk-3 beta in the other drug treatment groups were significantly higher than those in the control group (p0.05), and the levels of pser9-gsk-3 beta in the combined drug treatment group were also significantly higher than those in the single drug treatment group (p0.05). Conclusion: 1. After ovariectomy, the estrogen level in the systemic circulation of AD mice decreased with age, and the uterine morphology gradually atrophied with age. 2. After ovariectomy, the spatial learning and memory ability of AD mice gradually decreased with age. 3. After ovariectomy, the deposition of A beta in the brain and the formation of senile plaque of AD mice increased, which may be related to ovariectomy. The mechanism may be related to the abnormal GSK-3 beta activity caused by the decrease of ER beta content. 4. Drug treatment significantly improved the depressed and anxious behavior of ovariectomized AD mice, alleviated the decline of their spatial learning and memory ability, and the effect of combined treatment was significantly better than that of single drug treatment. 5. Drug treatment significantly increased the cortex and sea of ovariectomized AD mice. The content of estrogen receptor in horse area was significantly increased. 6. Drug treatment significantly increased the content of P ser9-GSK-3 beta in cerebral cortex and hippocampus of ovariectomized AD mice, and inhibited the activity of GSK-3 beta, and the effect of combined treatment was more significant.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.16
本文編號：2206934
[Abstract]:AIM: To study the dynamic changes of estrogen receptors (ERs) and glycogen synthase kinase 3 beta (GSK 3 beta) in the brain of mice with estrogen deficiency at different time points, and to observe the effect of valproic acid sodium salt (VPA) combined with estrogen on the estrogen in the brain of APP / PS1 castrated AD model mice. The results of this study will provide a new laboratory basis for further revealing the pathogenesis of AD and clinical research and development of multi-targeted drugs for AD. METHODS: This study was carried out in two parts. Part I: Selection of 3-month-old female APP/PS1. Forty double transgenic mice were randomly divided into sham operation group (AD-Sham group) and operation group (AD-OVX group) and wild type mice of the same age group (WT group) with 10 mice in each group. Part: One month after ovariectomy, dementia model mice were randomly divided into 6 groups: normal saline control group, VPA drug treatment group, E2 drug treatment group, Liq drug treatment group, VPA combined with E2 drug treatment group and VPA combined with Liq drug treatment group, with 8 mice in each group. The treatment group was given E2 drug 2.4ug/kg/d, the liq treatment group was given liq50ug/kg/d, the VPA and E2 combined treatment group were given VPA 30mg/kg/d and e22.4ug/kg/d, the VPA and liq combined treatment group were given VPA 30mg/kg/d and liq50ug/kg/d, and the saline treatment group was given the same amount of saline, except the liq was given intraperitoneally. After one month of treatment, the depression and anxiety behaviors of mice in each group were tested by elevated cross maze and open field test; the spatial learning and memory abilities of mice were tested by Morris water maze; the estrogen receptor content, GSK-3 beta activity and senile plaque were detected by Western blot and other techniques. Results: 1. the results of the first part of the experiment: OVX 1 month and 5 months group AD mice circulatory estrogen levels significantly decreased (p0.05); OVX 1 month group mice uterus did not significantly shrink, but OVX 5 months showed significant atrophy. Morris water maze showed that OVX 1 month later, compared with ad-sham group, ad-ovx mice found a platform escape potential. The incubation period and distance increased significantly (p0.05), and the number of crossing platforms decreased significantly (p0.05). with the prolongation of OVX time, to OVX 5 months later, the time of finding platforms and the distance of crossing platforms in ad-ovx group were further prolonged (p0.05), and the number of crossing platforms decreased accordingly (p0.05). immunofluorescence results showed that compared with ad-sham group, ad-ovx 1. The results of Western blot showed that compared with ad-sham group, the level of ER-beta protein in the brain of ad-ovx mice decreased progressively (p0.05); accordingly, the decrease of p-gsk-3 beta level in the brain of ad-ovx mice was consistent with that of normal mice and AD mice (p0.05). There was no significant difference in the total protein of GSK-3 beta between the two groups (p0.05). 2. Part 2: 1, Behavior 1) The results of the elevated maze test showed that the number of activities in the open arm of the mice in the combined drug treatment group was significantly increased (p0.05), and the duration of stay in the open arm of the combined drug treatment group was significantly longer than that of the other groups (p0.01). The ratio of activity times and residence time of open arms in the VPA and E2 combined treatment group were significantly increased (p0.01), and the liq treatment group was also significantly increased (p0.05); VPA and e2, VPA and liq combined treatment group compared with the VPA and E2 single drug treatment group were significantly increased (p0.05). There was no significant difference in the time and distance between the two groups (p0.05). under the covert platform experiment, the swimming time and distance of the mice in the other drug treatment groups were significantly shorter than those in the normal saline control group (p0.05), while the swimming time and distance of the combined drug treatment group were shorter (p0.05). Compared with the saline group, the number of crossing platform in the combined drug treatment group was significantly increased (p0.01), and the number of crossing platform in the single drug treatment group was also significantly increased (p0.05); compared with the combined drug treatment group, VPA treatment group and liq treatment group were significantly reduced (p0.05), E2 treatment group was significantly reduced (p0.01). uterine morphology and weight statistics of mice in each group showed that: The uterus morphology of the mice in the treatment group increased in different degrees. the results of statistical analysis showed that the weight of uterus in VPA group, liq group and control group had no significant change (p0.05); the weight of uterus in E2 group, VPA + E2 group, VPA + liq group and control group increased significantly (p0.01). The results of protein level experiment: (1) compared with the normal saline and estrogen receptor alpha group, the content of ER alpha protein in the cerebral cortex increased significantly in the combined drug treatment group (p0.01), and there was no significant difference in the single drug treatment group (p0.05). Compared with the control group, the level of ER-alpha protein in the cerebral hippocampus did not change significantly in the VPA treatment group (p0.05), but increased significantly in the other drug treatment groups (p0.05). in the comparison of ER-beta content, compared with the normal saline control group, the content of ER-beta in the cerebral cortex and hippocampus of the other drug treatment groups increased significantly (p0.05). (2) GSK-3 beta activity test: compared with the normal saline control group, there was no significant difference in the levels of GSK-3 beta total protein in the cerebral cortex and hippocampus of mice treated with other drugs alone (p0.05), and there was no significant difference in GSK-3 beta total protein between the combined drug treatment group and the normal saline control group (p0.05). The total protein level decreased significantly (p0.05), but there was no significant difference in the total protein level of GSK-3 beta in the hippocampus (p0.05). the phosphorylation level of the ninth locus marked the activity of GSK-3 beta. compared with the normal saline control group, the levels of pser9-gsk-3 beta in the VPA group, E2 group and liq group had no significant difference (p0.05), and the combined drug treatment group. The levels of pser9-gsk-3 beta in the hippocampus of each group were significantly higher than those in the normal saline group (p0.05). The levels of pser9-gsk-3 beta in the other drug treatment groups were significantly higher than those in the control group (p0.05), and the levels of pser9-gsk-3 beta in the combined drug treatment group were also significantly higher than those in the single drug treatment group (p0.05). Conclusion: 1. After ovariectomy, the estrogen level in the systemic circulation of AD mice decreased with age, and the uterine morphology gradually atrophied with age. 2. After ovariectomy, the spatial learning and memory ability of AD mice gradually decreased with age. 3. After ovariectomy, the deposition of A beta in the brain and the formation of senile plaque of AD mice increased, which may be related to ovariectomy. The mechanism may be related to the abnormal GSK-3 beta activity caused by the decrease of ER beta content. 4. Drug treatment significantly improved the depressed and anxious behavior of ovariectomized AD mice, alleviated the decline of their spatial learning and memory ability, and the effect of combined treatment was significantly better than that of single drug treatment. 5. Drug treatment significantly increased the cortex and sea of ovariectomized AD mice. The content of estrogen receptor in horse area was significantly increased. 6. Drug treatment significantly increased the content of P ser9-GSK-3 beta in cerebral cortex and hippocampus of ovariectomized AD mice, and inhibited the activity of GSK-3 beta, and the effect of combined treatment was more significant.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.16
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