黄芪甲甙对异氟醚引起神经元凋亡的作用及异氟醚认知功能的临床研究

发布时间：2018-05-24 01:15

本文选题：黄芪甲甙 + 麻醉　；参考：《南京医科大学》2016年博士论文

【摘要】：第一部分:黄芪甲甙对异氟醚麻醉后新生大鼠神经元凋亡的保护作用麻醉药在神经元生长的突触发生阶段对大脑有损害作用,是由于麻醉药是通过NMDA(N-甲基-D-天冬氨酸)受体和GABA(γ-氨基丁酸)受体产生麻醉作用的。这种神经毒性的易损期是在突触的发生阶段,也就是大脑快速生长阶段。这种损害会影响整个神经网络的生长,并且这种损害会影响到以后的行为和认知能力。这些研究结果使人们对全身麻醉药的神经毒性作用更加关注,特别是对小儿来说,他们的大脑正处于麻醉药的神经毒性的易损期。神经毒性主要是因为神经元的凋亡。关于麻醉引起的神经元的凋亡机制已经被广泛研究。一方面促凋亡蛋白的活化如Bax等会导致线粒体膜的损伤,caspases的活化会引起细胞的凋亡。另一方面,活性氧族(ROS)和活性氮族(RNS)等自由基的产生会导致脂质过氧化反应,这种反应会导致大脑的损害。这种自由基的损害也会引起炎症反应,更进一步放大了凋亡作用。相反,如超氧化物歧化酶这种抗氧化剂(SOD)能清除过量的自由基并且能减轻他们的毒性作用。黄芪甲甙是从传统中药黄芪中提纯而来的一种皂素。它在各种类型的细胞核组织中都显示出抗氧化应激和抗凋亡的作用。例如,它能通过抑制丝裂原活化蛋白激酶(MAPK)的活性来抑制肾组织的纤维化,它能减轻单侧输尿管梗阻和转化生长因子-β(TGF-β)引起的肾小管细胞的凋亡。另外,它能够通过抑制ROS的产生和抑制Bax的促凋亡途径对1-甲基-4-苯基-吡啶离子引起的神经毒性产生保护作用。黄芪甲甙还能通过抑制线粒体通透性转换孔的开放来减轻1-甲基-4-苯基吡啶离子引起的神经毒性。然而,它在麻醉药物引起的神经元的凋亡和神经毒性中的作用还没有被研究。为了明确黄芪甲甙是否对麻醉引起的发育中的大脑神经元凋亡有保护作用,我们研究了黄芪甲甙预处理对异氟醚引起的新生大鼠海马组织神经元凋亡的影响。测量海马组织和血清中氧化应激相关的酶的活性,使用酶联免疫吸附剂测定(ELISA)法测量血清中促炎性因子的水平。使用免疫印迹试验(Western blot)分析法测量NF-κB,caspase-3,BCL-2,磷酸化c-Jun氨基末端激酶(phosphorylated JNK),细胞外调节蛋白激酶(ERK),蛋白激酶B(AKT)的蛋白表达水平。我们的实验表明黄芪甲甙对异氟醚引起的神经元的凋亡有保护作用,并且证明了其潜在的机制。综上所述,我们的研究提供了新的实验证据,证明了黄芪甲甙对麻醉相关的神经毒性有保护作用。研究目的:探讨黄芪甲甙预处理能否减少异氟醚麻醉下大鼠神经细胞的凋亡及其可能的机制。研究方法:新生的大鼠随机分为5组,每组6只大鼠:组1:异氟醚组;组2-4:黄芪甲甙预处理(10mg/kg,40mg/kg,100mg/kg通过灌胃法给予黄芪甲甙的溶解液预处理3天)组;对照组:未麻醉组。测量海马组织和血清中氧化应激相关的酶的活性,使用ELISA法测量血清中促炎性因子的水平。使用Western blot 分析法测量 NF-κB,caspase-3,BCL-2,phosphorylated JNK,ERK,and AKT 的蛋白表达水平。不同组别间的结果采用单因素方差分析。P0.05表示差异有统计学意义。研究结果:1、海马CA1区神经元凋亡在对照组海马CA1区没有死亡的神经元细胞。而在异氟醚组显示缩小的细胞质和变性的细胞核的死亡的细胞数量明显增多。与之相比,黄芪甲甙组神经元细胞的死亡数量明显减少。我们进一步通过TUNEL分析法检测这些凋亡细胞。在对照组,海马CA1区的神经元很少被TUNEL法染色,而异氟醚组则明显增多(P0.01)。相对的,黄芪甲甙组(40和100mg/kg)神经元细胞的凋亡明显减少(P0.01)。2、大鼠血清和海马中SOD,iNOS,MDA,NO测量我们分离出大鼠血清和海马组织,测量SOD、诱导型一氧化氮合酶(iNOS)、一氧化氮(NO)和丙二醛(MDA)酶活性。在对照组,血清和海马CA1的MDA水平相对较低。异氟醚组则显著抑制SOD活性水平,但促进凋亡的iNOS、NO和MDA的水平增高。相对的,黄芪甲甙组显著地抑制了异氟醚引起的MDA、iNOS和NO的产生,而提高了 SOD的活性水平。3、血清中促炎性因子的测量我们测量一些主要的促炎性因子的水平,如肿瘤坏死因子α(TNF-α),白细胞介素6(IL-6)和白细胞介素1β(IL-1β)。在对照组,这些因子的水平相对较低。在异氟醚组,这些因子的水平明显增高。相对的,在黄芪甲甙组(低剂量和高剂量组),显著降低了这些促炎性因子的释放(P0.01)。4、炎性反应和抗凋亡信号通路的分析我们通过Western blot的方法测量了大鼠海马组织中NF-κB的活性。在对照组的蛋白样本中,NF-κB蛋白主要存在细胞质中,细胞核含量很低,所以NF-κB活性很低。而异氟醚组在海马组织中细胞核中的NF-κB水平升高。相对的,在黄芪甲甙组(低剂量和高剂量组),细胞核中NF-κB的水平降低。我们测量了不同组别中凋亡相关的蛋白表达水平。在对照组中,促凋亡标记蛋白,Caspase-3相对较低,而抗凋亡标记蛋白BCL-2则大量表达。异氟醚组Caspase-3则显著升高,而BCL-2蛋白水平显著降低。相对的,在黄芪甲甙组(低剂量和高剂量组),则明显抑制了异氟醚引起的caspase-3上调和BCL-2下调。相似的,异氟醚组糖原合成酶激酶-3β(GSK-3β)水平升高,Klotho和磷酸化Akt蛋白水平降低。而黄芪甲甙组(低剂量和高剂量组)则显著抑制了异氟醚引起的这些改变。我们观察了异氟醚组中JNK和ERK的活性水平,发现它们的磷酸化蛋白水平都增高。然而,在黄芪甲甙组(低剂量和高剂量组)则显著抑制了异氟醚引起的MAPK的活性水平。结论:我们的研究显示在发育中的大脑黄芪甲甙对异氟醚麻醉导致的神经元的凋亡有保护作用,这种保护作用和黄芪甲甙的抗氧化应激和抗炎性特性有关。氧化应激和炎症反应能激活NF-κB和JNK途径导致神经元细胞的凋亡,而黄芪甲甙能抑制这两种途径来产生神经保护作用。第二部分:异氟醚麻醉对小儿腹腔镜腹股沟斜疝修补术后认知功能的影响目的比较七氟醚与异氟醚吸入全身麻醉对小儿腹腔镜腹股沟斜疝修补术后认知功能的影响。方法选取我院2013年1月至2015年1月收治的行腹腔镜腹股沟修补术的患儿60例和正常儿童30例,将患儿随机分为异氟醚组、七氟醚组和正常儿童的对照组,每组各30例。异氟醚组、七氟醚组两组进行诱导麻醉后行腹腔镜腹股沟斜疝修补术,观察和记录异氟醚组、七氟醚组两组患儿术后苏醒及清醒时间,术后不良反应,手术过程中不同时间的心率、平均动脉压及3组小儿术前、术后3天和7天的韦氏智力量表评分。结果1异氟醚组患儿在手术开始、手术15min时的心率较七氟醚组低,但仍在正常心率范围内,差异有统计学意义,P0.05。异氟醚组与七氟醚组患儿在麻醉前、手术时、手术过程中及手术完毕后的平均动脉压无显著性差异,P0.05。七氟醚组患儿总的不良反应发生率(包括躁动)低于异氟醚组,差异有统计学意义(P0.05)。异氟醚组患儿的术后苏醒时间均比七氟醚组长,差异有统计学意义,P0.05。2术后3天异氟醚组术后认知功能障碍(POCD)的发生率为15%(26个儿童中有4个),异氟醚组在常识、词汇和数字广度项目评分明显低于对照组,术后7天差异无统计学意义。七氟醚组术后3天POCD的发生率为12%(25个儿童中有3个),词汇和数字广度项目评分明显低于对照组,术后7天差异无统计学意义。异氟醚组和七氟醚组术后3、7天比较差异无统计学意义。结论1吸入麻醉药(异氟醚和七氟醚)会引起腹腔镜腹股沟斜疝修补术儿童术后不同程度的POCD,这种影响是可逆性的,能在短期内恢复,影响时间是3天。2全凭吸入麻醉安全有效,七氟醚和异氟醚麻醉对术后认知功能的影响无差异。
[Abstract]:The first part: the protective effect of astragalin on the neuronal apoptosis of neonatal rats after isoflurane anaesthesia, the anesthetic effect on the brain at the synaptogenesis stage of neuron growth is due to the anesthetic effect of the anesthetic by NMDA (N- methyl -D- aspartic acid) receptor and GABA (gamma aminobutyric acid) receptor. This neurotoxicity is easy. The damage is at the stage of synapse, the rapid growth stage of the brain. This damage affects the growth of the whole neural network, and the damage affects future behavior and cognitive ability. These results make people more concerned about the neurotoxicity of general anesthetics, especially for children, their brains. The neurotoxicity of anesthetics is in a period of vulnerability to neurotoxicity. Neurotoxicity is mainly due to neuronal apoptosis. The mechanism of apoptosis induced by anesthesia has been widely studied. On the one hand, the activation of apoptotic proteins, such as Bax, can lead to mitochondrial membrane damage, and the activation of caspases causes cell apoptosis. On the other hand, the active oxygen family The formation of free radicals such as (ROS) and active nitrogen (RNS) can lead to lipid peroxidation, which causes brain damage. This free radical damage can also cause inflammation, further amplifying the effect of apoptosis. On the contrary, the superoxide dismutase, an antioxidant (SOD), can remove excessive free radicals and reduce them. The toxic effect of astragalin is a saponin purified from the traditional Chinese medicine Astragalus membranaceus. It shows anti oxidative stress and anti apoptosis in various types of nuclear tissue. For example, it can inhibit the fibrosis of the renal tissue by inhibiting the activity of mitogen activated protein kinase (MAPK). It can reduce unilateral ureteral stem. The apoptosis of renal tubular cells caused by hindrance and transforming growth factor beta (TGF- beta). In addition, it can protect the neurotoxicity caused by the 1- methyl -4- phenyl pyridine ion by inhibiting the production of ROS and inhibiting the apoptosis pathway of Bax. Astragaloside can also reduce the 1- methyl -4- by inhibiting the opening of the permeability transition pore of the grain body. The neurotoxicity caused by benzyl pyridine ions. However, its role in neuronal apoptosis and neurotoxicity induced by narcotic drugs has not yet been studied. In order to determine whether astragalin has protective effect on the apoptosis of brain neurons in the development of anaesthesia, we have studied the new birth of Astragaloside on isoflurane. The effects of neuronal apoptosis in the hippocampus of rats. The activity of enzymes related to oxidative stress in the hippocampus and serum was measured. The levels of proinflammatory factors in serum were measured by enzyme linked immunosorbent assay (ELISA). NF- kappa B, Caspase-3, BCL-2, and phosphorylated c-Jun amino terminal kinase (NF-) were measured by the immunoblotting test (Western blot) analysis. Phosphorylated JNK) the protein expression level of extracellular regulated protein kinase (ERK) and protein kinase B (AKT). Our experiment showed that astragalin had protective effects on the apoptosis of neurons induced by isoflurane and demonstrated its potential mechanism. The study aims: To investigate whether astragalin preconditioning can reduce the apoptosis and possible mechanism of nerve cells in rats under isoflurane anesthesia. The study method: the neonatal rats were randomly divided into 5 groups, 6 rats in each group: group 1: isoflurane group, group 2-4: astragaloside pretreatment (10mg/kg, 40mg/kg, 100mg/kg pass) In the control group, the control group was given the solution of astragaloside for 3 days. The control group: the activity of oxidative stress related enzymes in the hippocampus and serum was measured in the control group. The serum levels of proinflammatory factors were measured by the ELISA method. The protein table of NF- kappa B, Caspase-3, BCL-2, phosphorylated JNK, ERK, and AKT was measured by the Western blot analysis method. The results of different groups using single factor analysis of variance.P0.05 indicated that the difference was statistically significant. 1, the neuronal apoptosis in hippocampal CA1 region was not dead in the hippocampus CA1 area of the control group, but the number of cells in the isoflurane group that showed a reduced cytoplasm and denatured nucleus increased significantly. In contrast, the number of neuronal cell deaths in the astragalin group decreased significantly. We further detected these apoptotic cells by TUNEL analysis. In the control group, the neurons in the hippocampus CA1 region were rarely stained by TUNEL, while the isoflurane group increased significantly (P0.01). Relative, the apoptosis of the astragalin group (40 and 100mg/kg) neurons was obvious. Decrease (P0.01).2, SOD, iNOS, MDA, NO in rat serum and hippocampus, measure the serum and hippocampus of rats, measure SOD, inducible nitric oxide synthase (iNOS), nitric oxide (NO) and malondialdehyde (MDA) enzyme activity. In the control group, the level of MDA in the serum and hippocampus CA1 is relatively low. The levels of apoptotic iNOS, NO and MDA increased. Relative, astragalin group significantly inhibited the production of MDA, iNOS and NO induced by isoflurane, and increased the activity level.3 of SOD, and the levels of serum proinflammatory factors in serum were measured for some of the major proinflammatory factors, such as tumor necrosis factor alpha (TNF- alpha), interleukin 6 (IL-6), and Interleukin 1 beta (IL-1 beta). In the control group, the levels of these factors were relatively low. In the isoflurane group, the levels of these factors were significantly higher. Relative, in the astragalin group (low and high dose groups), the release of these proinflammatory factors (P0.01).4 was significantly reduced, and the analysis of the inflammatory response and anti apoptosis signaling pathway was carried out through Western The blot method measured the activity of NF- kappa B in the hippocampus of rats. In the sample of the control group, the NF- kappa B protein mainly existed in the cytoplasm, and the nucleus content was very low, so the activity of NF- kappa B was very low. And the level of NF- kappa B in the nucleus of the hippocampus in the isoflurane group was higher. The level of NF- kappa B in the nucleus was reduced. We measured the expression level of apoptosis related proteins in different groups. In the control group, the apoptotic marker protein, Caspase-3 was relatively low, and the anti apoptotic marker protein BCL-2 was expressed in large numbers. The Caspase-3 in the isoflurane group increased significantly, while the BCL-2 protein level decreased significantly. Low and high dose groups significantly inhibited the up regulation of Caspase-3 and down regulation of BCL-2 induced by isoflurane. Similarly, the level of glycogen synthetase kinase -3 beta (GSK-3 beta) in isoflurane group increased and the level of Klotho and phosphorylated Akt protein decreased, while the astragalin group (low and high dose groups) significantly inhibited these changes caused by isoflurane. We observed the activity levels of JNK and ERK in isoflurane group and found that their phosphorylated protein levels increased. However, the activity level of MAPK caused by isoflurane in the astragalin group (low dose and high dose group) was significantly inhibited. Conclusion: our study showed that the development of astragaloside in the development of isoflurane induced nerve induced by isoflurane anesthesia. The apoptosis has protective effect, which is related to the antioxidant stress and anti-inflammatory properties of Astragalus membranaceus. Oxidative stress and inflammatory response can activate NF- kappa B and JNK pathway to induce neuronal cell apoptosis, and Astragalus glucoside can inhibit these two pathways to produce neuroprotective effects. The second part: isoflurane anesthesia for children's abdominal cavity The effect of cognitive function after endoscopic inguinal hernia repair. Objective to compare the effects of sevoflurane and isoflurane on the cognitive function of laparoscopic inguinal hernia repair in children. Methods 60 children with laparoscopic inguinal repair and 30 cases of normal children were selected from January 2013 to January 2015. The machine was divided into isoflurane group, sevoflurane group and normal children's control group, 30 cases in each group. Two groups of isoflurane group and sevoflurane group underwent laparoscopic inguinal hernia repair after induction anesthesia. Observe and record isoflurane group and sevoflurane group, two groups of sevoflurane group after operation recovery and waking time, postoperative adverse reaction, and the heart of different time during the operation. Rate, mean arterial pressure and 3 groups of children before operation, 3 days and 7 days after operation. Results the heart rate of the 1 isoflurane group was lower than that of the sevoflurane group at 15min, but the difference was statistically significant in the normal heart rate range. The P0.05. isoflurane group and the sevoflurane group were in the operation and the operation process before the anesthesia. There was no significant difference in the average arterial pressure between the two groups. The incidence of total adverse reactions (including agitation) in the P0.05. sevoflurane group was lower than that in the isoflurane group (P0.05). The postoperative recovery time of the isoflurane group was significantly longer than the sevoflurane group, and the difference was statistically significant. 3 days after the operation, the isoflurane group was recognized after the operation. The incidence of POCD was 15% (4 in 26 children). In the isoflurane group, the scores of the vocabulary and digital breadth were significantly lower than those in the control group. There was no significant difference at 7 days after operation. The incidence of POCD in the sevoflurane group was 12% (3 of 25 children) 3 days after operation, and the vocabulary and digital breadth scores were significantly lower than those in the control group. There was no statistical difference at 7 days after operation. There was no significant difference in the 3,7 days after operation in the isoflurane group and the sevoflurane group. Conclusion 1 inhaled anesthetics (isoflurane and sevoflurane) can cause different degrees of POCD after laparoscopic inguinal hernia repair in children. This effect is reversible, can be recovered in the short term, and the time is 3 days.2. Inhalation anesthesia is safe and effective. Sevoflurane and isoflurane anesthesia have no difference in postoperative cognitive function.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R614

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