不同波形电针对老年大鼠肝叶部分切除术后认知功能障碍的影响

发布时间：2018-06-30 19:20

本文选题：电针 + 肝叶部分切除术　；参考：《西南医科大学》2017年硕士论文

【摘要】：背景与目的:术后认知功能障碍(postoperative cognitive dysfunction,POCD)是一种手术后发生在中枢神经系统常见的并发症,尤其在老年患者中发生率极高,其可导致患者出现长期性或永久性的认知功能损害,生活自理能力的下降,更严重时甚至丧失。值得重视的是:POCD的发生率还与患者术后远期死亡率成正相关,给家庭及社会带来较大的负担。国内外学者在POCD的发生机制及防治方面做了大量的工作,但其具体机制目前仍未完全阐明,也无理想的防治方法。研究认为患者经手术创伤后,可以诱发大脑出现局部自限性的炎症或全身性炎症(systemic inflammatory),后者是一种级联式的瀑布样炎症反应,最终能导致POCD的发生及发展。小胶质细胞是一种中枢神经系统(Central Nervous System,CNS)固有的先天性免疫细胞,多年来被认为参与了神经退化疾病及神经炎症的病理过程。越来越多的证据表明创伤、外周炎症等因素刺激小胶质细胞,导致其激活后,其能释放诸多神经毒性物质,包括:肿瘤坏死因子(TNF)-α,白细胞介素(IL)-6,一氧化氮(NO)和活性氧(ROS)。这些毒性物质相互促进作用,导致了神经细胞凋亡及损伤。小胶质细胞涉及的细胞毒性因子也广泛参与了多种组织器官缺血再灌注损伤、炎症等多种病理生理过程,但其是否参与肝叶部分切除术所致的POCD的发生尚未见较多报道。针灸是我国传统医学的重要治疗方法之一,具有操作简单,明确的功效,经济安全,无副作用等优点,已在国内外医疗领域吸引了人们的广泛关注。此外,根据病人的具体情况,可以通过选择适当的穴位进行个性化治疗以提高疗效。电针(electroacupuncture,EA)刺激是在传统针灸的基础上、通过改变电刺激参数生效。事实证明,电针对心脏,肾脏和脑等器官损伤有预防和治疗作用。它可以改善学习和记忆功能,调节免疫系统、抗炎、抗氧化、抗凋亡等。我们前期研究已证实EA能通过抑制小胶质细胞激活,降低氧化损伤以及炎症反应,防治肢体缺血再灌注后的脑损伤。电针参数,通常包括脉冲输出的波形、频率和强度,是一个能明显影响电针治疗作用,并能改变其作用机制的重要因素。电针参数对治疗效果有很大影响,能够导致机体出现的不同的治疗反应。因此,不同的疾病应该选择不同的参数以达到最好的疗效。在先前的有关EA对中枢神经系统影响的研究中,主要是使用的疏密波,少数研究则使用连续波或不连续波。然而,使用EA防治POCD的研究是有限的,不同波形电针对POCD的影响及其机制目前尚不清楚。因此,本实验拟采用老年大鼠行肝叶部分切除术所致的术后认知功能障碍模型,探讨小胶质细胞在肝叶部分切除术所致POCD中的作用,同时研究不同波形EA防治老年大鼠肝叶部分切除术所致POCD的作用与小胶质细胞的关系。本研究的完成有望为POCD发生及EA防治POCD机制注入新的内容:一方面为防治POCD提供重要的可供干预的作用靶点,有利于临床针对小胶质细胞开发防治POCD的药物;另一方面可为不同波形EA保护效应提供理论基础。方法:120只Wistar雄性老年大鼠,随机分为5组(n=24):假手术组(S组)、手术组(O组)、电针连续波组(CW组)、电针疏密波组(DW组)、电针间断波组(IW组)。建立肝叶部分切除模型,电针组于术前电针刺激“百会”穴和“大椎”穴,留针30 min,每日1次,连续治疗3d。用Morris水迷宫评定大鼠术后1d、2d及5d认知功能;于术后2d,取脑组织,用免疫组化法观察海马小胶质细胞(MG)活化情况;采用Western blot法检测MG标志物Iba-1的表达水平;测定活性氧(ROS)、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、白细胞介素(IL)-1β、IL-6和肿瘤坏死因子(TNF)-α含量;通过原位末端标记(TUNEL)法观察脑细胞凋亡,计算凋亡指数。结果:(1)认知功能:与S组比较,O组及电针各组大鼠术后1d、2d及5d的潜伏期和游泳距离显著延长,穿越原平台次数减少,差异有统计学意义(p0.05);与O组比较,CW、DW、IW三电针组潜伏期和游泳距离显著缩短,穿越原平台次数增加,差异有统计学意义(p0.05);CW、DW、IW组之间比较,潜伏期、游泳距离及穿越原平台次数的差异均有统计学意义(p0.05),且DW组IW组CW组。(2)海马组织中小胶质细胞变化:与S组比较,O组及电针各组Iba-1阳性细胞数、小胶质细胞激活百分比及Iba-1蛋白表达增加,差异有统计学意义(p0.05);与O组比较,CW、DW、IW三组Iba-1阳性细胞数、小胶质细胞激活百分比及Iba-1蛋白表达量明显降低,差异有统计学意义(p0.05);CW、DW、IW组之间比较,以上指标差异均有统计学意义(p0.05)。DW最为显著,IW次之,CW降幅较小。(3)海马组织中IL-1β、IL-6及TNF-α浓度:与S组比较,O组及电针各组IL-1β、IL-6及TNF-α含量增加,有统计学差异(p0.05);与O组比较,CW、DW、IW三组IL-1β、IL-6及TNF-α含量降低,差异也有统计学意义(p0.05);CW、DW、IW组之间比较,以上指标差异均有统计学意义(p0.05)。DW降低最明显,IW次之,CW最小。(4)海马组织中ROS、MDA含量和SOD活性:与S组比较,O组及电针各组ROS、MDA含量明显增加、SOD活性明显降低,差异有统计学意义(p0.05);与O组比较,CW、DW、IW三组ROS、MDA含量均降低、SOD活性均增强(p0.05);CW、DW、IW组之间比较,以上指标差异均有统计学意义(p0.05)。DW降低最明显,IW次之,CW最小。(5)海马神经细胞凋亡:S组可见极少量凋亡细胞;O组可见大量凋亡细胞。与S组比较,O组及电针各组凋亡指数显著增加(p0.05);与O组比较,CW、DW、IW三组凋亡指数均降低,差异有统计学意义(p0.05);CW、DW、IW组之间比较,以上指标差异均有统计学意义(p0.05)。DW降低最明显,IW次之,CW最小。结论:1.肝叶部分切除术可以导致老年大鼠出现认知功能障碍,诱发小胶质细胞激活,促使海马组织炎性因子、氧化应激损伤及神经细胞凋亡增加;2.不同波形电针刺激“百会”,“大椎”穴均可改善老年大鼠术后认知功能障碍,以疏密波最佳,间断波次之,连续波最差;3.电针预处理改善老年大鼠POCD的机制可能与抑制小胶质细胞激活,减轻炎症反应、氧化应激损伤及神经细胞凋亡有关。
[Abstract]:Background and purpose: postoperative cognitive dysfunction (POCD) is a common complication occurring in the central nervous system after operation, especially in the elderly patients, which can lead to chronic or permanent cognitive impairment, the decline of self-care ability, and even more serious time. The loss. It is worth noting that the incidence of POCD is also positively related to the long-term mortality of the patients, bringing a greater burden to the family and society. Scholars at home and abroad have done a lot of work on the mechanism and prevention of POCD, but the specific mechanisms are still not fully elucidated and no ideal methods of prevention and control. After trauma, it can induce local self limiting inflammation or systemic inflammation (systemic inflammatory) in the brain, which is a cascade type of waterfall like inflammation that ultimately leads to the occurrence and development of POCD. Microglia is an inherent immune cell of the central nervous system (Central Nervous System, CNS) for many years. It is considered to be involved in the pathological process of neurodegenerative diseases and neuro inflammation. More and more evidence suggests that trauma, peripheral inflammation and other factors stimulate microglia, which can release many neurotoxic substances, including tumor necrosis factor (TNF) - alpha, IL -6, nitric oxide (NO) and reactive oxygen species (ROS). The mutual promotion of toxic substances leads to the apoptosis and damage of nerve cells. The cytotoxic factors involved in microglia are also widely involved in a variety of pathophysiological processes, such as ischemia-reperfusion injury and inflammation, but the involvement of POCD in partial hepatectomy has not yet been reported. One of the important treatment methods of traditional Chinese medicine has the advantages of simple operation, clear effect, economic safety and no side effect. It has attracted people's attention in the medical field at home and abroad. In addition, according to the specific situation of the patients, we can improve the curative effect by selecting appropriate acupoints for individualized treatment to improve the curative effect. (electroacupunctu Re, EA) stimulation is on the basis of traditional acupuncture and moxibustion by changing the parameters of electrical stimulation. In fact, it has been proved to have preventive and therapeutic effects on injury of heart, kidney and brain. It can improve learning and memory function, regulate immune system, anti-inflammatory, antioxidant, anti withering, and so on. Our previous study has confirmed that EA can inhibit microglia by inhibiting microglia. Cell activation, reducing oxidative damage and inflammatory reaction to prevent the brain damage after ischemia and reperfusion. Electroacupuncture Parameters, usually including pulse output waveform, frequency and intensity, are an important factor that can significantly affect the therapeutic effect of electroacupuncture and can change the mechanism of its action. The electroacupuncture parameters have a great influence on the therapeutic effect and can lead to the effect of the treatment. Different therapeutic responses to the body appear. Therefore, different diseases should choose different parameters to achieve the best effect. In previous studies on the effects of EA on the central nervous system, the main use of the dense wave, and the use of continuous or discontinuous waves in a few studies, but the use of EA to prevent and control POCD is limited. The effect of Electroacupuncture on POCD and its mechanism are still not clear. Therefore, this experiment is to use the model of postoperative cognitive dysfunction caused by partial hepatectomy in old rats, to explore the role of microglia in POCD caused by partial hepatectomy, and to study the prevention and treatment of partial hepatectomy by different wave forms of EA in old rats. The relationship between the role of POCD and microglia. The completion of this study is expected to infuse new contents for the occurrence of POCD and the mechanism of EA for the prevention and treatment of POCD. On the one hand, it provides an important target for intervention for the prevention and control of POCD, and is beneficial to the development of the drug for the development of the microglia for the prevention of POCD; on the other hand, it can provide the reason for the different waveform EA protection effects. Methods: 120 Wistar male aged rats were randomly divided into 5 groups (n=24): the sham operation group (group S), the operation group (group O), the electroacupuncture continuous wave group (group CW), the electroacupuncture group (group DW) and the intermittent wave group (group IW). The partial excision model of the hepatic lobe was established. The electroacupuncture group stimulated the "Baihui" point and the "Dazhui" point by Electroacupuncture before the operation, and the needle left 30 min, 1 daily, and daily 1. 3D. Morris water maze was used to evaluate the cognitive function of 1D, 2D and 5D in rats after operation. The activation of hippocampal microglia (MG) was observed by 2D and immunohistochemical method after operation, and the expression level of Iba-1 on MG marker was detected by Western blot method, and the activity of active oxygen (ROS), malondialdehyde and superoxide dismutase were measured by Western blot method. The content of interleukin (IL) -1 beta, IL-6 and tumor necrosis factor (TNF) - alpha, apoptosis and apoptosis index were observed by in situ terminal labeling (TUNEL) method. Results: (1) cognitive function: compared with the S group, the latency and swimming distance of 1D, 2D and 5D in the O group and the electroacupuncture group were significantly longer than those in the S group. Study significance (P0.05); compared with group O, the latency and swimming distance of CW, DW, IW three were significantly shortened, the number of crossing the original platform increased, the difference was statistically significant (P0.05); the difference between CW, DW, IW group, latency, swimming distance and the frequency of crossing the original platform were statistically significant (P0.05), and DW group IW group. (2) medium and small hippocampal tissue. Compared with the S group, the number of Iba-1 positive cells, the percentage of microglia activation and the expression of Iba-1 protein increased in group O and electroacupuncture, and the difference was statistically significant (P0.05). Compared with the O group, the number of Iba-1 positive cells in CW, DW, IW three groups, the percentage of activation of microglia and the expression of Iba-1 protein decreased significantly, and the difference was statistically significant. P0.05, CW, DW, and IW were statistically significant (P0.05).DW, IW time and CW decline. (3) the concentration of IL-1 beta, IL-6 and TNF- alpha in the hippocampus: compared with the S group, there was a statistical difference between the groups and the electroacupuncture groups. And TNF- alpha content decreased, the difference also had statistical significance (P0.05); CW, DW, IW group, the above index difference was statistically significant (P0.05).DW decrease most obvious, IW times, CW minimum. (4) ROS, MDA content and SOD activity in the hippocampus: compared with the group and Electroacupuncture groups Statistical significance (P0.05); compared with group O, CW, DW, IW three groups ROS, MDA content decreased, SOD activity increased (P0.05); CW, DW, there was a significant difference between the IW groups. (5) apoptosis of the hippocampal neurons: a large number of apoptotic cells in the group; and a large number of apoptotic cells. Compared with group S, apoptosis index of group O and electroacupuncture increased significantly (P0.05). Compared with group O, the apoptotic index in CW, DW, IW three groups decreased, the difference was statistically significant (P0.05); CW, DW, IW group had statistical significance (P0.05) was the most significant difference between the groups of CW, DW, IW. Conclusion: 1. partial hepatectomy can lead to old age. In rats, cognitive dysfunction, induced microglia activation, induced inflammatory factors in the hippocampus, oxidative stress injury and neuronal apoptosis increased; 2. different waveform electroacupuncture stimulation of "Baihui" and "Da Zhui" can improve the cognitive impairment of old rats after operation, with the best density wave, discontinuous wave, and the worst continuous wave; 3. electroacupuncture. The mechanism of pretreatment in improving POCD in aged rats may be related to inhibiting microglial activation, reducing inflammatory reaction, oxidative stress injury and neuronal apoptosis.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R657.3

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