七氟烷致小鼠发育期大脑神经毒性的机制研究

发布时间：2018-06-25 23:29

本文选题：七氟烷 + 认知功能　；参考：《天津医科大学》2017年博士论文

【摘要】：七氟烷(Sevoflurane)是新生儿及儿童临床上最常应用的全身麻醉药(General Anesthetics)[1,2],其对小儿发育期大脑的神经毒性受到广泛关注,但具体机制尚不清楚。研究表明,儿童期多次暴露于全身麻醉药物更易造成其发育期大脑的神经毒性,从而影响其远期学习记忆能力(Learning ability)[3-5],而Tau蛋白磷酸化在其认知功能损伤的发生发展中具有重要的作用[6-9]。近年来,有研究指出,非微管结合Tau蛋白(Microtubule-unbound Tau,MUT),可能是Tau蛋白代谢异常(磷酸化及异常聚集)的早期标志,而能量及Nuak1(Nuak family SNF1-like kinase 1)对其有调控作用[10,11]。本课题以Nuak1/Tau信号通路为切入点,深入研究七氟烷多次麻醉对幼年及成年小鼠认知功能及神经损伤的影响,并探讨其分子机制,同时寻求相关脑保护方法。实验一:七氟烷多次麻醉对不同年龄小鼠认知功能改变及Tau蛋白表达的影响目的:七氟烷多次麻醉可致幼年小鼠远期认知功能障碍,Tau蛋白过度磷酸化可造成神经损伤,而非微管结合Tau蛋白可能是Tau蛋白早期代谢的标志。本实验拟探讨七氟烷多次麻醉对幼年(P6)及成年(P60)小鼠远期认知功能改变及不同类型Tau蛋白及其磷酸化位点表达的影响。方法:出生6 d(P6)及出生60 d(P60)小鼠各52只随机分为4组(n=26/组):幼年对照组(P6+Control)、幼年七氟烷组(P6+Sevoflurane)、成年对照组(P60+Control)、成年七氟烷组(P60+Sevoflurane)。其中七氟烷组给予3%七氟烷+60%O2处理,每天2 h,连续3 d,对照组只给予60%O2处理,每天2 h,连续3 d。七氟烷连续处理后22 d,对不同组小鼠(P30,P84)进行Morris水迷宫实验检测其认知功能;七氟烷连续处理后0 d(P8,P62),取小鼠海马及皮层组织,应用ELISA检测total Tau表达、RT-PCR检测Tau m RNA表达、Western blot检测total Tau(Tau46),T22(Tau蛋白早期聚合物,可溶性低聚体,oligomers)Tau-PS202/PT205(PHF-Tau),Tau-PS356(R4)表达、微管结合实验检测不同类型Tau蛋白(微管结合Tau蛋白及非微管结合Tau蛋白)表达、荧光染色检测Tau蛋白早期聚集(T22)、串联质谱分析检测Tau蛋白不同磷酸化位点表达。结果:1.七氟烷多次麻醉可致幼年小鼠远期认知功能损伤,而对成年小鼠没有影响;2.幼年小鼠皮层和海马组织中Tau m RNA及total Tau蛋白表达均高于成年小鼠;3.幼年小鼠皮层及海马组织T22及Tau-PS356表达明显高于成年小鼠,七氟烷处理可使幼年小鼠皮层及海马Tau-PS202/PT205表达增加,而成年小鼠则没有差异;4.幼年小鼠海马及皮层主要为非微管结合Tau蛋白,而成年小鼠主要为微管结合Tau蛋白;5.幼年小鼠脑片中可在其大脑皮层发现Tau蛋白早期聚集(可溶性低聚体,T22),而成年小鼠脑片不见聚集;6.七氟烷多次麻醉可致幼年小鼠皮层大部分Tau蛋白位点磷酸化增强,而对成年小鼠影响不大。结论:幼年小鼠与成年小鼠相比,其发育期大脑更易遭受到七氟烷多次麻醉的打击,造成Tau蛋白多位点磷酸化增加,从而影响其远期认知功能,其机制与非微管结合Tau蛋白表达密切相关。实验二:Nuak1在七氟烷多次麻醉所致发育期小鼠大脑神经毒性中的关键作用目的:2016年,一项新研究指出,Nuak1(Nuak family DNF1-like kinase 1,又叫做AMPK related protein 5,ARK5)可通过选择性磷酸化位于Tau蛋白重复序列(R4)上的Ser356位点,从而调节Tau蛋白代谢。本实验拟通过探讨Nuak1对Tau蛋白的调控作用,通过应用Nuak1特异性阻滞剂HTH-01-015,拟证明Nuak1在七氟烷多次麻醉所致发育期小鼠大脑神经毒性中的关键作用。方法:出生6 d(P6)及出生60 d(P60)小鼠各12只随机分为4组(n=6/组):幼年对照组(P6+Control)、幼年七氟烷组(P6+Sevoflurane)、成年对照组(P60+Control)、成年七氟烷组(P60+Sevoflurane),相关处理同实验一。多次麻醉后0 d(P8,P62)后立即处死小鼠,提取其海马及皮层组织,应用RT-PCR及Western blot检测不同年龄组小鼠大脑皮层及海马Nuak1基因及蛋白表达、应用串联质谱(MS/MS)分析Nuak1不同磷酸化位点表达;另选80只P6小鼠,随机分为4组(n=20/组):对照+溶剂组(Control+Vehicle)、七氟烷+溶剂组(Sevoflurane+Vehicle)、对照+Nuak1阻滞剂组(Control+HTH-01-015)、七氟烷+Nuak1阻滞剂组(Sevoflurane+HTH-01-015)。七氟烷及氧气处理同上,所有P6小鼠于七氟烷麻醉及氧气处理前30 min,腹腔注射(i.p.)Nuak1特异性阻滞剂HTH-01-015(10 mg/kg)或者溶剂(生理盐水+DMSO)100μL/次,连续注射3 d,并于七氟烷处理后22 d(P30)通过Morris水迷宫实验检测认知功能;于七氟烷及氧气处理后0 d,取皮层及海马组织,通过Western blot实验检测Nuak1、total Tau(Tau46)、T22、Tau-PS356及Tau-PS202/PT205表达、通过免疫荧光染色检测T22蛋白聚集情况、通过微管结合实验检测不同类型Tau蛋白(微管结合Tau蛋白及非微管结合Tau蛋白)表达。结果:1.幼年小鼠大脑皮层及海马Nuak1蛋白表达明显高于成年小鼠,而Nuak1基因表达(m RNA)与成年小鼠相比没有统计学意义;2.串联质谱分析得出,幼年小鼠Nuak1只有2个磷酸化位点(Nuak1-PS389,Nuak1-PS446)表达,并且幼年小鼠皮层组织Nuak1磷酸化表达明显低于成年小鼠;3.给予Nuak1特异性阻滞剂HTH-01-015后,对于幼年小鼠,七氟烷多次处理并没有造成其远期认知功能损伤;4.给予Nuak1特异性阻滞剂后,幼年小鼠大脑皮层及海马Nuak1及total Tau表达没有差异,而Tau-PS356、Tau-PS262/PT205表达与对照+溶剂组相比均降低,且七氟烷处理并不能使其增加;5.给予Nuak1阻滞剂后,幼年小鼠皮层及海马中的非微管结合Tau蛋白明显减少,Tau蛋白早期聚集明显降低。结论:Nuak1通过选择性磷酸化位于重复序列R4上的Tau-PS356,使Tau蛋白与微管解离,从而增加非微管结合Tau蛋白含量,七氟烷多次麻醉可刺激非微管结合Tau蛋白,从而激活GSK3β,正反馈形成超磷酸化,从而产生神经毒性,以致远期认知功能障碍。而Nuak1含量受自身磷酸化调节,其在脑中的蛋白表达与其自身磷酸化成反比。实验三:能量不足(Energy deprivation)在七氟烷多次麻醉致小鼠发育期大脑神经毒性中的作用目的:研究指出,能量不足(Energy deprivation)和氧化应激(Oxidative stress)可能是导致Tau蛋白磷酸化聚集的主要原因[12]。本实验通过应用维生素K2(一种能量补充剂),拟证明能量在七氟烷所致小鼠发育期大脑神经毒性中的作用。方法:出生6 d(P6)及出生60 d(P60)小鼠各12只随机分为4组(n=6/组):幼年对照组(P6+Control)、幼年七氟烷组(P6+Sevoflurane)、成年对照组(P60+Control)、成年七氟烷组(P60+Sevoflurane),相关处理同实验一。多次麻醉后0d(P8,P62)处死小鼠,提取皮层组织,利用ATP检测试剂盒检测不同年龄组小鼠ATP表达;另选80只P6小鼠,随机分为4组(n=20/组):对照+玉米油组(Control+Corn oil)、七氟烷+玉米油组(Sevoflurane+Corn oil)、对照+维生素K2组(Control+Vitamin K2)、七氟烷+维生素K2组(Sevoflurane+Vitamin K2)。七氟烷组小鼠给予3%七氟烷+60%O2处理,每天2 h,连续3 d,对照组只给予60%O2处理,每天2 h,连续3 d。所有P6小鼠于七氟烷麻醉及氧气处理前30 min,腹腔注射(i.p.)Vitamin K2(100 mg/kg)或者玉米油100μL/次,连续注射3 d,并于七氟烷处理后22 d(P30)通过Morris水迷宫实验检测认知功能;于七氟烷及氧气处理后0 d,取皮层及海马组织,通过ELISA检测total Tau表达、通过Western blot实验检测Nuak1、total Tau(Tau46)、T22、Tau-PS356,Tau-PS202/PT205表达、通过免疫荧光染色检测Tau蛋白聚集情况、通过微管结合实验检测不同类型Tau表达。结果:1.幼年小鼠皮层的ATP表达明显低于成年小鼠,且七氟烷刺激可使ATP明显降低;2.给予Vitamin K2后,对于P6小鼠,七氟烷多次处理并没有造成其远期认知功能损伤;3.给予Vitamin K2后,P6小鼠大脑皮层及海马total Tau蛋白表达没有差异,而Nuak1、Tau-PS356、Tau-PS262/PT205表达与对照+玉米油组相比均降低,且七氟烷处理并不能使其增加;4.给予Vitamin K2后,幼年小鼠皮层及海马中的非微管结合Tau蛋白明显减少,Tau蛋白早期聚集(T22)明显消失。结论:幼年小鼠大脑皮层及海马能量表达明显低于成年小鼠,而能量不足(能量剥夺)本身可以激活AMPK,使得Nuak1表达增加,从而使得Tau蛋白,特别是非微管结合Tau蛋白的含量增加,造成神经脆弱性(Neuronal vulnerability),形成Tau蛋白早期聚集,由此,在七氟烷多次麻醉的刺激下,Tau蛋白大量脱微管,磷酸化甚至超磷酸化,从而造成神经发育损伤,影响远期认知及学习功能。小结1.七氟烷多次麻醉可造成幼年小鼠远期认知功能障碍,对成年小鼠没有影响;2.幼年小鼠发育期大脑中主要表达非微管结合Tau蛋白,而成年小鼠大脑主要表达微管结合Tau蛋白;3.幼年小鼠发育期大脑皮层中Tau蛋白早期聚集造成神经脆弱性,在七氟烷多次麻醉下形成Tau蛋白大量位点超磷酸化,从而造成神经损伤;4.Nuak1通过选择性磷酸化Tau-PS356以调节Tau蛋白代谢,在七氟烷多次麻醉致小鼠发育期大脑神经毒性中起关键作用;5.幼年小鼠大脑ATP含量远远低于成年小鼠,能量严重不足;6.能量不足可使Nuak1磷酸化降低,从而使Nuak1表达增加,并造成幼年小鼠发育期大脑神经脆弱性,可能是七氟烷多次麻醉致小鼠发育期大脑神经损伤的根本原因。
[Abstract]:Seven fluorane (Sevoflurane) is the most commonly used general anesthetic (General Anesthetics) [1,2] for newborns and children. The neurotoxicity of the brain is widely concerned in the development of children, but the specific mechanism is not clear. It affects its long term learning and memory (Learning ability) [3-5], and Tau protein phosphorylation plays an important role in the occurrence and development of cognitive impairment. In recent years, studies have shown that non microtubules combined with Tau protein (Microtubule-unbound Tau, MUT), can be the early stage of metabolic abnormalities of Tau proteins (phosphorylation and abnormal aggregation). Mark, energy and Nuak1 (Nuak family SNF1-like kinase 1) have a regulatory effect on [10,11]. this topic with Nuak1/Tau signaling pathway as the breakthrough point, in-depth study of the effects of seven halothane multiple anaesthesia on cognitive function and nerve damage in young and adult mice, and explore its molecular mechanism, and seek related brain protection methods. Experiment 1: Seven The effects of multiple anaesthesia on cognitive function changes and Tau protein expression in mice of different ages: seven fluoroalkanes can cause long-term cognitive impairment in young mice. Excessive phosphorylation of Tau protein can cause nerve damage, while non microtubule combined with Tau protein may be a marker of early metabolism of Tau protein. This experiment is to discuss seven fluoroalkanes. The effect of secondary anesthesia on the long-term cognitive function changes and the expression of different types of Tau protein and its phosphorylation sites in young (P6) and adult (P60) mice. Methods: 52 mice born 6 d (P6) and 60 d (P60) mice were randomly divided into 4 groups (n=26/ group): juvenile control group (P6+ Control), juvenile seven fluorane group (P6+Sevoflurane), adult control group (P60+Control), In the adult seven fluorane group (P60+Sevoflurane), the seven halothane group was given 3% seven fluorothane +60%O2 treatment, 2 h a day and 3 D continuously. The control group was given only 60%O2 treatment, 2 h a day, and 22 D continuously after 3 d. seven Fluoroalkane continuous treatment, and the cognitive function was detected in the Morris water maze test for different groups of mice (P30, P84); 0 d (seven fluorothane) after continuous treatment. The expression of total Tau was detected by ELISA, and Tau m RNA expression was detected by RT-PCR, Western blot detected total Tau (Tau46). The expression of non microtubule combined with Tau protein, the early aggregation of Tau protein (T22) was detected by fluorescence staining, and the expression of different phosphorylation sites of Tau protein was detected by tandem mass spectrometry. Results: 1. seven halothane multiple anaesthesia could cause long-term cognitive impairment in young mice, but no effect on adult mice; 2. the Tau m RNA and total T in the cortex and hippocampus of young mice. The expression of Au protein was higher than that of adult mice; the expression of T22 and Tau-PS356 in the cortex and hippocampus of 3. young mice was significantly higher than that of adult mice. Seven halothane treatment could increase the expression of Tau-PS202/PT205 in the cortex and hippocampus of young mice, but there was no difference in adult mice. 4. of the hippocampus and cortex of young mice were mainly non microtubules combined with Tau protein, and adult mice were small. The rats were mainly microtubules combined with Tau protein; 5. young mice brain slices could find early aggregation of Tau protein (soluble oligomer, T22) in its cerebral cortex, while the brain slices of adult mice were not clustered; 6. seven fluoroalkanes could increase the phosphorylation of most of the Tau protein loci in young mice, but little effect on adult mice. Conclusion: young young mice are small. Compared with adult mice, the brain of the adult mice is more susceptible to the attack of seven halothane anaesthesia, resulting in the increase of Tau protein multipoint phosphorylation, which affects its long-term cognitive function. The mechanism is closely related to the non microtubule binding of Tau protein. Experiment two: Nuak1 in the brain neurotoxicity induced by seven fluoroalkanes during multiple anaesthesia. In 2016, a new study indicated that Nuak1 (Nuak family DNF1-like kinase 1, also called AMPK related protein 5, ARK5) can selectively phosphorylate the Ser356 sites on the Tau protein repeat sequence (R4), thus regulating the metabolism of proteins. Nuak1 specific blocker HTH-01-015 was used to prove the key role of Nuak1 in the brain neurotoxicity of mice induced by seven halothane anesthesia. Methods: 12 mice born 6 d (P6) and 60 d (P60) mice were randomly divided into 4 groups (n=6/ group): juvenile control group (P6+ Control), juvenile seven halothane group (P6+Sevoflurane), adult control group (P60+Contr) OL), adult seven fluorane group (P60+Sevoflurane), related treatment with experimental one. After multiple anesthesia 0 d (P8, P62) immediately after the death of mice, extract the hippocampus and cortical tissue, the use of RT-PCR and Western blot to detect the cerebral cortex and hippocampus Nuak1 gene and protein expression in different age groups of mice, using tandem mass spectrometry (MS/MS) to analyze Nuak1 phosphorylation 80 P6 mice were randomly divided into 4 groups (group n=20/): control + solvent group (Control+Vehicle), seven fluorane + solvent group (Sevoflurane+Vehicle), control +Nuak1 blocker group (Control+HTH-01-015), seven fluorothane +Nuak1 blocker group (Sevoflurane+ HTH-01-015). Seven halothane and oxygen treatment, all P6 mice were anesthetized with seven halothane and anaesthesia. 30 min before oxygen treatment, intraperitoneal injection (i.p.) Nuak1 specific blocker HTH-01-015 (10 mg/kg) or solvent (physiological saline +DMSO) 100 mu L/ time, continuous injection of 3 D, and 22 D (P30) after seven fluorane treated by Morris water maze test to detect cognitive function; seven fluorane and oxygen treated 0 d, cortex and hippocampus tissue, through The expression of Nuak1, total Tau (Tau46), T22, Tau-PS356 and Tau-PS202/PT205 was detected. The aggregation of T22 protein was detected by immunofluorescence staining. The expression of different types Tau protein (microtubule binding Tau protein and non microtubule binding Tau protein) was detected by microtubule binding assay. The expression of Nuak1 protein in the cerebral cortex and hippocampus of 1. young mice was obvious. Compared with adult mice, the expression of Nuak1 gene (m RNA) was not statistically significant compared with adult mice. 2. tandem mass spectrometry analysis showed that there were only 2 phosphorylation sites (Nuak1-PS389, Nuak1-PS446) expression of Nuak1 in young mice, and the Nuak1 phosphorylation of the cortical tissue of young mice was significantly lower than that of adult mice; 3. gave Nuak1 specific blocker HTH. After -01-015, for young mice, seven Fluoroalkane multiple treatments did not cause their long-term cognitive impairment. 4. after Nuak1 specific blockers were given, the expression of Nuak1 and total Tau in the cerebral cortex and hippocampus of young mice was not different, while Tau-PS356, Tau-PS262/PT205 expression was lower than that of the control + solvent group, and seven fluorothane treatment did not make it possible. It increased; 5. after the Nuak1 blocker was given, the non microtubule binding Tau protein in the cortex and hippocampus of the young mice decreased obviously, and the early aggregation of Tau protein decreased obviously. Conclusion: Nuak1 dissociates the Tau protein from the microtubule by selectively phosphorylation of Tau-PS356 on the repeated sequence R4, and increases the content of the non microtubule binding Tau protein, and the seven Fluoroalkane multiple times. Anesthesia stimulates non microtubule binding Tau protein, thus activating GSK3 beta, positive feedback forms hyper phosphorylation, resulting in neurotoxicity, resulting in long-term cognitive dysfunction. The Nuak1 content is regulated by autophosphorylation and its protein expression in the brain is inversely proportional to its own phosphorylation. Three: Energy deprivation (Energy deprivation) is more than seven fluoroalkanes. The role of secondary anesthesia in the brain neurotoxicity of mice during development: the study indicated that Energy deprivation and oxidative stress (Oxidative stress) may be the main cause of the aggregation of Tau protein phosphorylation. This experiment was used to prove that the energy was induced by seven halothane in mice by using vitamin K2 (a energy supplement). Methods: 6 d (P6) and 60 d (P60) mice were randomly divided into 4 groups (n=6/ group): young control group (P6+Control), juvenile seven fluorane group (P6+Sevoflurane), adult control group (P60+Control), adult seven fluorane group (P60+Sevoflurane), related treatment and experiment one. 0d (P8, P62) after multiple anesthesia were executed. In mice, the cortex tissues were extracted and the ATP detection kit was used to detect ATP expression in different age groups. 80 P6 mice were randomly divided into 4 groups (group n=20/): control + corn oil group (Control+Corn oil), seven fluorane + corn oil group (Sevoflurane+Corn oil), control + vitrein K2 group (Control+Vitamin K2), seven halothane + vitamin K2 group Amin K2). Seven fluorane group mice were treated with 3% seven fluorane +60%O2 treatment, 2 h a day, 3 D. The control group was given only 60%O2 treatment, 2 h per day, 3 d. in 3 d. and 30 min before seven Fluoroalkane. The cognitive function was detected by the Morris water maze test. The expression of total Tau was detected by ELISA and 0 d after seven Fluoroalkane and oxygen treatment. The expression of Nuak1, total Tau (Tau46), T22, expression were detected by Western blot experiment. The aggregation of protein was detected by immunofluorescence staining and combined with microtubule through immunofluorescence staining. The expression of different types of Tau was detected in the experiment. Results: the expression of ATP in the cortex of 1. young mice was significantly lower than that of adult mice, and the stimulation of seven halothane decreased the ATP obviously. 2. to P6 mice after Vitamin K2, the multiple treatment of seven Fluoroalkane did not cause its long-term cognitive impairment; 3. Vitamin K2, P6 mice cerebral cortex and hippocampus total Tau. There was no difference in protein expression, but the expression of Nuak1, Tau-PS356 and Tau-PS262/PT205 decreased compared with the control + corn oil group, and seven halothane treatment did not increase it. 4. after Vitamin K2, the non microtubule binding Tau protein in the cortex and hippocampus of young mice decreased obviously, and the early aggregation of Tau egg white (T22) disappeared. Conclusion: the brain of young mice. The energy expression in the cortex and hippocampus is obviously lower than that of the adult mice, and the energy deprivation (energy deprivation) itself can activate AMPK and increase the expression of Nuak1, thus increasing the content of Tau protein, especially the non microtubule combined with Tau protein, causing nervous vulnerability (Neuronal vulnerability), forming the early aggregation of the Tau protein, thus, in seven Fluoroalkane multiple times. Under the stimulation of anesthesia, Tau protein has a large number of microtubules, phosphorylation and even hyper phosphorylation, which can cause neurodevelopmental damage and affect long-term cognitive and learning functions. 1. seven halothane multiple anaesthesia can cause long-term cognitive impairment in young mice and no effect on adult mice; 2. the main expression of non microtubule binding in the brain of young mice. Tau protein, while the adult mouse brain mainly expresses microtubule combined with Tau protein; 3. early accumulation of Tau protein in the cerebral cortex of young mice causes nerve fragility, resulting in the formation of a large number of Tau protein sites hyper phosphorylation under seven halothane anaesthesia, resulting in nerve damage; 4.Nuak1 regulates the Tau protein passage through selective phosphorylation of Tau-PS356. It plays a key role in the brain neurotoxicity of mice during the development of seven fluorothane. 5. the ATP content in the brain of 5. young mice is much lower than that of adult mice, and the energy is seriously insufficient; the deficiency of 6. energy can reduce the phosphorylation of Nuak1, thus increasing the expression of Nuak1 and causing the nervous fragility of the brain in the developmental stage of the young rats, which may be more than seven Fluoroalkane. The underlying cause of brain damage during developmental period is secondary anesthesia.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R614

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