线粒体分裂融合在铝致大鼠认知功能障碍中的作用

发布时间：2018-07-04 18:10

本文选题：铝 + 认知功能　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:通过体内实验探讨线粒体分裂融合在铝致大鼠认知功能障碍的作用。方法:180只清洁级健康雄性SD大鼠,采用随机数字表法按体质量随机分为5组,即空白组、溶剂对照组、低、中、高剂量组,每组12只。溶剂对照组、低、中、高剂量组分别予剂量为0.41、0.81、1.62 mg/kg体质量的麦芽酚铝溶液,溶剂对照组予等量生理盐水,空白组不施加任何干预措施,腹腔注射染毒,隔日注射。大鼠分为3批,第一批染毒一个月,第二批染毒二个月,第三批染毒三个月。染毒结束后,采用Morris水迷宫实验测定大鼠空间学习记忆能力,采用石墨炉法测定大鼠海马铝含量,采用透射电镜观察线粒体超微机构,采用化学比色法测定海马组织线粒体酶活性,采用Western-blot检测海马组织CoxⅣ、Drp1、Fis1、Opa1、Mfn1、Mfn2、CaN和S-drp1(s637)蛋白的相对表达水平。结果:1.Morris水迷宫实验检测结果:(1)定位航行实验逃避潜伏期重复测量方差分析各批大鼠训练时间逃避潜伏期在染铝剂量与训练时间上交互作用不存在统计学意义(F1=1.648,P=0.125;F2=0.986,P=0.453;F3=0.763,P=0.664)。三批老鼠通过训练,逃避潜伏期均随训练时间的延长而缩短(F1=182.462,P0.001;F2=307.703,P0.001;F3=110.248,P0.001)。(2)空间探索试验目标象限停留时间空间探索实验大鼠目标象限停留时间染铝剂量与染铝时间交互作用有统计学意义(F=4.853,P0.001),随着染毒时间和染毒剂量的增加,大鼠目标象限停留时间均减少(P0.05)。(3)空间探索实验穿越平台次数随着染毒剂量和染毒时间的增加,大鼠穿越平台次数减少。大鼠穿越平台次数在染铝剂量与染铝时间的交互作用无统计学意义(F=0.409,P=0.913)。2.石墨炉法测定大鼠海马脑铝含量:对大鼠脑铝水平进行铝暴露时间与剂量的析因方差分析,结果显示各铝暴露时间组间脑铝差异有统计学意义(f=9.009;p0.05),后续进行铝暴露时间组两两比较发现与铝暴露1个月组相比,2个月组和3个月组脑铝水平明显上升(p0.05)。大鼠脑铝结果在铝暴露时间与剂量上并无交互作用(f=1.097;p0.05)。3.透射电镜线粒体超微结构结果:染铝3个月大鼠高剂量组海马组织线粒体与空白组、溶剂对照组相比多出现肿胀,内棘大部分排列紊乱,有些内部出现空泡化,线粒体棘溶解,线粒体变大变圆随时有破裂的迹象。4.化学比色法测定海马组织线粒体酶活性结果:(1)对三批次各剂量组大鼠海马na+-k+atp酶活性表达量析因分析,结果表明na+-k+atp酶活性表达量在染铝剂量与染铝时间交互作用有统计学意义(f=2.675,p=0.012)。因此随着染毒剂量的增加,na+-k+atp酶活性降低(p0.05),仅中剂量和高剂量组染毒三个月与染毒两个月差别无统计学意义,各组比较差异均具有统计学意义。(2)对三批次各剂量组大鼠海马ca2+-mg2+atp酶活性表达量析因分析,结果表明ca2+-mg2+atp酶活性表达量在染铝剂量与染铝时间交互作用有统计学意义(f=3.665,p0.001)。随着染毒剂量的增加,ca2+-mg2+atp酶活性降低(p0.05)。5.western-blot检测海马组织coxⅣ、drp1、fis1、opa1、mfn1、mfn2、can和s-drp1(s637)蛋白的相对表达水平结果:(1)coxⅣ对三批次各剂量组大鼠海马coxⅣ表达量析因分析,结果表明coxⅣ蛋白表达量在染铝剂量与染铝时间交互作用有统计学意义(f=2.11,p0.05),随着染毒剂量的增加,蛋白表达量降低(p0.05)。(2)drp1对三批次各剂量组大鼠海马drp1表达量析因分析,结果表明drp1蛋白表达量在染铝剂量与染铝时间交互作用有统计学意义(f=4.306,p0.001)。随着染毒剂量的增加,蛋白表达量增加(p0.05)。(3)fis1对三批次各剂量组大鼠海马fis1表达量析因分析,结果表明fis1蛋白表达量在染铝剂量与染铝时间交互作用无统计学意义(f=1.168,p=0.330)。高剂量组fis1蛋白表达量分别比空白组、溶剂对照组和低剂量组蛋白表达量增多0.19倍、0.16倍和0.14倍。(4)opa1、mfn1、mfn2对三批次各剂量组大鼠海马opa1表达量析因分析,结果表明opa1、mfn1、mfn2蛋白表达量在染铝剂量与染铝时间交互作用无统计学意义(f=1.942,p=0.066)、(f=1.012,p=0.435)、(f=0.255,p=0.978)。高剂量组大鼠opa1蛋白表达量比空白组、溶剂对照组、低剂量组、中剂量分别增加1.12倍、1.07倍、0.39倍、0.22倍;高剂量组大鼠Mfn1蛋白表达量比空白组、溶剂对照组、低剂量组、中剂量分别增加0.71倍、0.70倍、0.47倍、0.21倍;高剂量组Mfn2蛋白表达量分别比空白组、溶剂对照组和低剂量组蛋白表达量增多0.41倍、0.37倍和0.2倍。(5)S-drp1(s637)对三批次各剂量组大鼠海马s-Drp1(637)表达量析因分析,结果表明s-Drp1(s637)蛋白表达量在染铝剂量与染铝时间交互作用有统计学意义(F=2.477,P=0.019)随着染毒剂量的增加,蛋白表达量降低,染毒三个月时高剂量组蛋白表达量相比溶剂对照组、低剂量、中剂量分别降低了67.5%、51.8%、21.2%。(6)对三批次各剂量组大鼠海马CaN表达量析因分析,结果表明CaN蛋白表达量在染铝剂量与染铝时间交互作用有统计学意义(F=2.904,P=0.047)。随着染毒剂量的增加,蛋白表达量增加,染毒三个月时高剂量组蛋白表达量相比溶剂对照组、低剂量、中剂量分别增加了0.64倍、0.31倍、0.10倍。结论:1.亚慢性染铝可导致大鼠学习记忆能力损伤,致大鼠认知功能障碍,且存在剂量及时间依赖性。2.铝暴露可致大鼠海马线粒体损伤,线粒体功能下降,且存在时间剂量依赖性。3.铝暴露可打破大鼠海马线粒体分裂融合平衡,且以打破线粒体分裂为主,具有时间剂量依赖性。综上所述,铝可能通过打破线粒体分裂融合平衡进而引起线粒体损伤、功能下降,最终会导致大鼠认知功能障碍。
[Abstract]:Objective: To investigate the effect of mitochondrial mitotic fusion on cognitive dysfunction induced by aluminum in rats. Methods: 180 healthy male SD rats were randomly divided into 5 groups by random number table method according to body mass, namely, blank group, solvent control group, low, middle and high dose group, with 12 rats in each group. The group of low, middle and high dose groups were given respectively. The dose of 0.41,0.81,1.62 mg/kg body mass of Maltol aluminum solution, the solvent control group was given the same amount of physiological saline, the blank group did not exert any intervention measures, intraperitoneal injection and daily injection. The rats were divided into 3 batches, the first batch was poisoned for one month, the second batches were poisoned for two months, and the third batches were poisoned for three months. After the end of the poisoning, the Morris water maze was used. The spatial learning and memory ability of rats was measured. The content of aluminum in the hippocampus of rats was measured by graphite furnace method. The mitochondrial ultrastructure was observed by transmission electron microscopy. The activity of mitochondrial enzyme in the hippocampus was measured by chemical colorimetry. The relative expression of Cox IV, Drp1, Fis1, Opa1, Mfn1, Mfn2, CaN and S-drp1 (s637) protein in the hippocampus was detected by Western-blot. Results: results: the results of 1.Morris water maze test: (1) the analysis of variance analysis of the escape incubation period of the navigation experiment, there is no statistical significance (F1=1.648, P=0.125; F2= 0.986, P=0.453; F3=0.763, P=0.664) in the training time of the training time of each batch of rats in the escape incubation period of the training time of each batch of rats (P=0.125, F3=0.763, P=0.664). The incubation period shortened with the prolongation of the training time (F1=182.462, P0.001; F2=307.703, P0.001; F3=110.248, P0.001). (2) space exploration test target quadrant time space exploration of experimental rats' target quadrant time, the interaction between aluminum dye and aluminum dyeing time was statistically significant (F=4.853, P0.001), with the time of exposure and exposure. The residence time of the target quadrant of rats decreased (P0.05). (3) the number of crossing platform in rats decreased with the increase of exposure dose and time. The interaction between the number of rat crossing platform and the interaction between the dose of aluminum dye and the time of aluminum dyeing was not statistically significant (F=0.409, P=0.913).2. graphite furnace method The content of aluminum in the hippocampus of rats was determined by the analysis of variance analysis of aluminum exposure time and dose of aluminum exposure in rats. The results showed that there was a significant difference between the aluminum exposure time group and the aluminum exposure group (f=9.009; P0.05). Compared with the aluminum exposure group 22, the aluminum exposure was compared with the 1 months of aluminum exposure group, and the 2 months group and 3 month group of brain al water were clear. There was a significant increase (P0.05). There was no interaction between the aluminum exposure time and the dose of aluminum exposure (f=1.097; P0.05). The ultrastructural results of mitochondrial ultrastructure of.3. transmission electron microscopy: the hippocampus mitochondria and the blank group in the high dose group of 3 months of aluminum infected rats, the swelling, the disorder of the most of the internal spines, and the vacuolation in some of the internal spines were found in the high dose group. The mitochondrial spines dissolved, the mitochondria became large and round and there were signs of rupture at any time..4. chemical colorimetry was used to determine the results of mitochondrial enzyme activity in the hippocampus: (1) analysis of the expression of na+-k+atp enzyme activity in the hippocampus of three batches of rats. The results showed that the interaction of na+-k+atp enzyme activity expression at the dose of aluminum and the time of aluminum dyeing was statistically significant. Meaning (f=2.675, p=0.012). Therefore, with the increase of dose, the activity of na+-k+atp enzyme decreased (P0.05). There was no statistically significant difference between the middle dose and high dose group for three months and two months. (2) the analysis of ca2+-mg2+atp enzyme activity expression in the hippocampus of three batches of rats The results showed that the interaction of the activity of ca2+-mg2+atp enzyme activity was statistically significant (f=3.665, p0.001). With the increase of dose, the activity of ca2+-mg2+atp enzyme decreased (P0.05).5.western-blot to detect the relative expression level of Cox IV, drp1, FIS1, OPA1, mfn1, Mfn2, Mfn2, drp1, and.5.western-blot in the hippocampus. (1) analysis of Cox IV expression in hippocampus of rats in each dose group of Cox IV to three batches. The results showed that the expression of Cox IV protein expression was statistically significant (f=2.11, P0.05) at the dose of aluminum dye and aluminum dyeing time (f=2.11, P0.05). The expression of protein decreased with the increase of dose (P0.05). (2) the expression of drp1 in the hippocampus of each dose group of three batches of drp1 was analyzed by drp1 The results showed that the interaction of drp1 protein expression and aluminum dyeing time was statistically significant (f=4.306, p0.001). As the dose increased, the protein expression increased (P0.05). (3) the analysis of FIS1 expression in hippocampal FIS1 of FIS1 to three batches of rats showed that the expression of FIS1 protein was in the dose of aluminum and aluminum. The time interaction was not statistically significant (f=1.168, p=0.330). The expression of FIS1 protein in the high dose group was 0.19 times more than that in the blank group, the expression of the protein in the solvent control group and the low dose group increased by 0.19 times, 0.16 times and 0.14 times. (4) OPA1, mfn1, Mfn2 were analyzed for the expression of OPA1 in the hippocampus of three batches of rats. The results showed that the expression of OPA1, mfn1, Mfn2 protein was expressed. There was no statistical significance (f=1.942, p=0.066), (f=1.012, p=0.435), (f=1.012, p=0.435), (f=0.255, p=0.978). The expression of OPA1 protein in the high dose group was 1.12 times more, 1.07 times, 0.39 times, 0.22 times than that in the blank group, the low dose group and the low dose group respectively. The expression of Mfn1 protein in the high dose group was more than that of the blank group. In the solvent control group, the low dose group increased 0.71 times, 0.70 times, 0.47 times, 0.21 times respectively. The expression of Mfn2 protein in the high dose group was 0.41 times, 0.37 times and 0.2 times more than that in the blank group, 0.37 times and 0.2 times in the solvent control group and the low dose group. (5) the expression of the s-Drp1 (637) in the hippocampus of the three batches of rats was analyzed, the result of the analysis of the results of the expression of the s-Drp1 (637) in the hippocampus of three batches of rats. The results showed that the expression of s-Drp1 (s637) protein expression was statistically significant (F=2.477, P=0.019), with the increase of the dose, the protein expression decreased, and the high dose of protein expression was compared with the solvent control group at three months. The low dose and middle dose decreased by 67.5%, 51.8%, and 21.2%. (6) to three batches, respectively. The analysis of CaN expression in the hippocampus of dose group showed that the expression of CaN protein expression was statistically significant (F=2.904, P=0.047) in the dose of aluminum dyed and aluminum dye (F=2.904, P=0.047). With the increase of dose, the protein expression increased, and the high dose of protein expression was compared with the solvent control group at three months, and the low dose and middle dose increased respectively. The addition of 0.64 times, 0.31 times, and 0.10 times. Conclusion: 1. subchronic aluminum dyed aluminum can lead to the impairment of learning and memory ability in rats, and induce cognitive dysfunction in rats. There is a dose and time dependent.2. aluminum exposure that can cause damage to the mitochondria of the hippocampus in rats and the decrease of mitochondrial function, and the presence of time dependent.3. aluminum exposure can break the mitochondria of the hippocampus of rats. In summary, aluminum may cause mitochondrial damage by breaking mitochondrial fission and fusion, resulting in mitochondrial damage and decreased function, which eventually leads to cognitive dysfunction in rats.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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