氢气对脓毒症小鼠脑损伤的保护作用及其相关机制

发布时间：2018-05-10 23:03

本文选题：氢气 + 脓毒症　；参考：《天津医科大学》2015年硕士论文

【摘要】：背景:脓毒症是指由感染或高度可疑感染因素引起的全身炎症反应综合征(systemic inflammatory response syndrome,SRIS),是创伤、手术后的常见并发症,以及重症监护病房内非心脏病患者死亡的主要原因[1,2]。研究发现,脓毒症患者出现中枢神经系统并发症早于其他系统,且脓毒症患者伴脑功能障碍时死亡率相对明显提高[3]。此外,9~71%脓毒症存活者伴有不同程度的认知功能障碍。因此,改善脓毒症脑功能障碍对脓毒症的治疗和预后均具有重要意义。氢气作为一种新型的气体信号分子,具有明显抗炎、抗氧化、抗凋亡及信号调节作用[4]。前期研究发现,2%氢气吸入可明显提高脓毒症小鼠的存活率,并减轻心、肺和肾等重要器官损伤[5,6]。本文拟在前期研究的基础上,采用盲肠结扎穿孔法诱导脓毒症小鼠脑损伤,探讨2%氢气吸入对脓毒症小鼠脑损伤的保护作用及其相关机制,为其临床应用提供理论依据。目的:观察2%氢气吸入对脓毒症小鼠的的存活率和认知功能的影响,并探讨其对脓毒症诱导的脑损伤的保护作用和相关机制。方法:成年雄性ICR小鼠,重20~25 g,随机数字表法分为假手术组(Sham组)、假手术+氢气组(Sham+H2组)、脓毒症组(CLP组)和脓毒症+氢气组(CLP+H2组)。采用盲肠结扎穿孔法(CLP)诱导脓毒症小鼠脑损伤。氢气治疗组小鼠在建模后1 h和6 h时分别吸入2%氢气1 h。于假手术或CLP后7 d内观察各组小鼠的存活情况。于假手术或CLP后3~14 d通过Y-迷宫、条件恐惧实验及Morris水迷宫检测各组小鼠的短期工作记忆、长期恐惧记忆能力及学习和空间识别记忆能力。在建模后1 h和6 h时给予氢气吸入,通过氢微电极分别检测各组小鼠氢气吸入后和停止氢气吸入后不同时间点动脉血、静脉血及脑组织氢气浓度。于假手术或CLP后24 h时,通过尼氏染色和TUNEL染色观察脑组织病理改变和神经元凋亡情况;通过伊文氏蓝法和脑组织含水量的测定观察脑组织血脑屏障的通透性;通过Elisa或比色法检测血浆和海马组织中TNF-α、IL-1β、HMGB1、IL-10的浓度,SOD和CAT的活性及MDA和8-iso-PGF2α的水平;通过Western Blot检测总Nrf2、核Nrf2及HO-1的蛋白表达情况;通过免疫荧光染色观察各组小鼠海马组织中Nrf2的表达及核转位情况;通过免疫组化染色观察各组小鼠海马组织中HO-1的定位和表达。结果:2%氢气明显提高脓毒症小鼠7 d内的存活率(P0.05)。在适应和训练阶段,各组小鼠的僵直时间百分比无明显差异(P㧐0.05);于假手术或CLP后3~14 d,氢气明显提高脓毒症小鼠的交替百分比(P0.01);氢气显著提高脓毒症小鼠的僵直时间百分比(P0.01);在隐蔽站台试验中,氢气明显缩短脓毒症小鼠的潜伏期(P0.001),游泳速度无明显差异(P㧐0.05);在空间探索实验中,氢气显著提高脓毒症小鼠在目标象限的时间百分比和穿过站台次数(P0.001)。在假手术或CLP后1 h和6 h氢气吸入后不同时间点,Sham+H2组和CLP+H2组的动脉血、静脉血和脑组织中氢气浓度均较CLP组均明显提高(P0.05),而在停止吸入氢气后不同时间点,CLP组和CLP+H2组动脉血氢气浓度低于Sham+H2组(P0.05)。于假手术后24 h时,Sham组和Sham+H2组小鼠海马CA1区正常椎体神经元数量无明显差异,基本无神经元凋亡。于CLP后24 h时,与Sham组相比,CLP组和CLP+H2组小鼠海马CA1区正常椎体神经元数量减少,凋亡神经元数量增多,EB含量和脑组织含水量增多(P0.05);与CLP组相比,CLP+H2组小鼠海马CA1区正常椎体神经元数量明显增多,凋亡神经元明显减少,EB含量和脑组织含水量明显减少(P0.05)。于假手术或CLP后24 h时,与Sham组相比,CLP组和CLP+H2组血浆和海马组织匀浆中TNF-α、IL-1β、HMGB1和IL-10水平均增高,SOD和CAT的活力均降低,MDA和8-iso-PGF2α水平均增高,总Nrf2表达增多,部分转位至胞核中,HO-1表达和HO-1阳性细胞数均增多(P0.05);与CLP组相比,CLP+H2组小鼠血浆和海马组织匀浆中TNF-α、IL-1β和HMGB1水平均明显降低,IL-10水平明显增高,SOD和CAT的活力均明显提高,MDA和8-iso-PGF2α水平均明显降低,总Nrf2表达明显增多,细胞核内Nrf2明显增多,HO-1表达和HO-1阳性细胞数均显著增多(P0.05)。Sham组和Sham+H2组各项指标无明显差异(P㧐0.05)。结论:2%氢气吸入可明显提高脓毒症小鼠的存活率,改善脓毒症小鼠的短期工作记忆和长期恐惧记忆能力,还可提高其学习和空间识别记忆能力,同时还可明显改善脓毒症小鼠脑组织病理损伤,减少神经元凋亡及减轻血脑屏障的破坏,其保护作用可能通过激活Nrf2/HO-1通路,来抑制炎症反应,提高抗氧化酶的活性,降低氧化产物的水平,从而减轻脑组织损伤。
[Abstract]:Background: sepsis is a systemic inflammatory response syndrome (systemic inflammatory response syndrome, SRIS) caused by infection or highly suspicious infection factors. It is a trauma, a common complication after surgery, and a major cause of death in the ICU, and the main cause of death in the intensive care unit ([1,2].) found that the central nervous system of sepsis patients The systemic complications are earlier than other systems, and the mortality of sepsis patients with brain dysfunction is significantly increased by [3]., and 9~71% sepsis survivors are associated with different degrees of cognitive impairment. Therefore, the improvement of sepsis brain dysfunction is of great significance for the treatment and prognosis of sepsis. Hydrogen is a new type of gas letter. [4]., which has obvious anti-inflammatory, antioxidation, anti apoptosis and signal regulation, found that 2% hydrogen inhalation can obviously improve the survival rate of sepsis mice and reduce the damage of important organs, such as heart, lung and kidney, [5,6].. On the basis of earlier study, the cecum ligation perforation method was used to induce the brain damage of sepsis mice. 2% the protective effect of hydrogen inhalation on the brain injury of sepsis mice and its related mechanism provide the theoretical basis for its clinical application. Objective: To observe the effect of 2% hydrogen inhalation on the survival rate and cognitive function of sepsis mice, and to explore the protective effect and mechanism of its effect on the brain injury induced by sepsis. Methods: adult male ICR mice, 20~25 g, randomly divided into sham operation group (group Sham), sham operation + hydrogen group (group Sham+H2), sepsis group (CLP group) and sepsis + hydrogen group (CLP+H2 group). The cecum ligation perforation (CLP) was used to induce the brain injury of sepsis mice. The mice in the hydrogen treatment group inhaled 2% hydrogen and 1 h. respectively at 1 h and 6 h after modeling, and 7 d and 7 d after the sham operation or CLP. The survival of mice in each group was observed. The short-term working memory, long-term fear memory ability and learning and spatial recognition and memory ability were detected by the Y- maze, the condition fear experiment and the Morris water maze after the sham operation or the CLP 3~14 D. The hydrogen gas was inhaled at 1 h and 6 h after modeling, and each group was detected by hydrogen microelectrodes. The hydrogen concentration of arterial blood, venous blood and brain tissue at different time points after hydrogen inhalation and stopping hydrogen inhalation. The pathological changes of brain tissue and neuron apoptosis were observed by Nissl's staining and TUNEL staining at 24 h after sham operation or after CLP, and the blood brain barrier of brain tissue was observed through the evens blue method and the water content of brain tissue. Permeability; the concentration of TNF- alpha, IL-1 beta, HMGB1, IL-10, the activity of SOD and CAT and the level of MDA and 8-iso-PGF2 alpha in the plasma and hippocampus were detected by Elisa or colorimetric method. The expression of the total Nrf2, the expression of the protein and the protein expression of the total Nrf2 were detected by Western Blot; the expression and nuclear transposition situation in the hippocampus of each group were observed by immunofluorescence staining. The localization and expression of HO-1 in the hippocampus of each group was observed by immunohistochemical staining. Results: 2% hydrogen was significantly increased in the survival rate of 7 d in sepsis mice (P0.05). There was no significant difference (P? 0.05) in the percentage of the stiffness of the mice in the adaptation and training stages (P? 0.05). In the sham operation or after CLP 3~14 D, the hydrogen was significantly increased in sepsis mice. Alternating percentage (P0.01); hydrogen significantly increased the percentage of stiff time in sepsis mice (P0.01); in a hidden platform test, hydrogen significantly shortened the incubation period of sepsis mice (P0.001), and there was no significant difference in swimming speed (P? 0.05). In the space exploration experiment, hydrogen significantly increased the percentage of time in the target quadrant of sepsis mice. The concentration of hydrogen in the arterial blood, the venous blood and the brain tissue of the Sham+H2 group and the CLP+H2 group increased significantly (P0.05) at different time points after the sham operation or 1 h and 6 h hydrogen after CLP inhalation (P0.05). The hydrogen concentration in the arterial blood of the CLP group and the CLP+H2 group was lower than that of the Sham+H2 group (P0.) after stopping inhalation of hydrogen (P0.) (P0.) (P0.) was lower than that of the Sham+H2 group (P0.) (P0.) (P0.05). 05). At 24 h after the sham operation, there was no significant difference in the number of normal vertebra neurons in the hippocampus CA1 region of the Sham group and the Sham+H2 group. At 24 h after CLP, the number of normal vertebral neurons in the CA1 region of CLP and CLP+H2 mice decreased, the number of apoptotic neurons increased, the EB content and the water content in the brain tissue increased. Compared with the CLP group, the number of normal vertebral neurons in the hippocampus CA1 area of the CLP+H2 group was significantly increased, the apoptotic neurons decreased obviously, and the content of EB and the water content of the brain decreased significantly (P0.05). Compared with the 24 h in the sham operation or the CLP after the CLP, the TNF- a, the beta, and the levels of the plasma and hippocampal homogenate of the CLP and CLP+H2 groups were compared with the Sham group. Both the activity of SOD and CAT decreased, the level of MDA and 8-iso-PGF2 alpha increased, the expression of total Nrf2 increased, and the number of HO-1 expression and HO-1 positive cells increased (P0.05). Compared with the CLP group, TNF- alpha in the plasma and hippocampal homogenates of the CLP+H2 group were significantly lower. The activity of MDA and CAT increased obviously, the level of MDA and 8-iso-PGF2 alpha decreased obviously, the expression of total Nrf2 increased obviously, the Nrf2 increased in the nucleus, and the number of HO-1 expression and HO-1 positive cells increased significantly (P0.05) in.Sham group and Sham+H2 group (P? 0.05). Conclusion: 2% hydrogen inhalation can obviously improve the survival of sepsis mice. Rate, improve the short-term working memory and long-term fear memory ability of sepsis, and improve the ability of learning and spatial recognition and memory. At the same time, it can obviously improve the pathological damage of brain tissue in septic mice, reduce the apoptosis of neurons and reduce the destruction of blood brain barrier. The protective effect may be inhibited by activating the Nrf2/HO-1 pathway to inhibit inflammation. It can enhance the activity of antioxidant enzymes and reduce the level of oxidation products, thereby reducing brain tissue damage.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R459.7

【共引文献】