本文選題：氫氣 + 膿毒癥�。� 參考：《天津醫(yī)科大學(xué)》2015年碩士論文

【摘要】：背景:膿毒癥是指由感染或高度可疑感染因素引起的全身炎癥反應(yīng)綜合征(systemic inflammatory response syndrome,SRIS),是創(chuàng)傷、手術(shù)后的常見并發(fā)癥,以及重癥監(jiān)護(hù)病房?jī)?nèi)非心臟病患者死亡的主要原因[1,2]。研究發(fā)現(xiàn),膿毒癥患者出現(xiàn)中樞神經(jīng)系統(tǒng)并發(fā)癥早于其他系統(tǒng),且膿毒癥患者伴腦功能障礙時(shí)死亡率相對(duì)明顯提高[3]。此外,9~71%膿毒癥存活者伴有不同程度的認(rèn)知功能障礙。因此,改善膿毒癥腦功能障礙對(duì)膿毒癥的治療和預(yù)后均具有重要意義。氫氣作為一種新型的氣體信號(hào)分子,具有明顯抗炎、抗氧化、抗凋亡及信號(hào)調(diào)節(jié)作用[4]。前期研究發(fā)現(xiàn),2%氫氣吸入可明顯提高膿毒癥小鼠的存活率,并減輕心、肺和腎等重要器官損傷[5,6]。本文擬在前期研究的基礎(chǔ)上,采用盲腸結(jié)扎穿孔法誘導(dǎo)膿毒癥小鼠腦損傷,探討2%氫氣吸入對(duì)膿毒癥小鼠腦損傷的保護(hù)作用及其相關(guān)機(jī)制,為其臨床應(yīng)用提供理論依據(jù)。目的:觀察2%氫氣吸入對(duì)膿毒癥小鼠的的存活率和認(rèn)知功能的影響,并探討其對(duì)膿毒癥誘導(dǎo)的腦損傷的保護(hù)作用和相關(guān)機(jī)制。方法:成年雄性ICR小鼠,重20~25 g,隨機(jī)數(shù)字表法分為假手術(shù)組(Sham組)、假手術(shù)+氫氣組(Sham+H2組)、膿毒癥組(CLP組)和膿毒癥+氫氣組(CLP+H2組)。采用盲腸結(jié)扎穿孔法(CLP)誘導(dǎo)膿毒癥小鼠腦損傷。氫氣治療組小鼠在建模后1 h和6 h時(shí)分別吸入2%氫氣1 h。于假手術(shù)或CLP后7 d內(nèi)觀察各組小鼠的存活情況。于假手術(shù)或CLP后3~14 d通過Y-迷宮、條件恐懼實(shí)驗(yàn)及Morris水迷宮檢測(cè)各組小鼠的短期工作記憶、長(zhǎng)期恐懼記憶能力及學(xué)習(xí)和空間識(shí)別記憶能力。在建模后1 h和6 h時(shí)給予氫氣吸入,通過氫微電極分別檢測(cè)各組小鼠氫氣吸入后和停止氫氣吸入后不同時(shí)間點(diǎn)動(dòng)脈血、靜脈血及腦組織氫氣濃度。于假手術(shù)或CLP后24 h時(shí),通過尼氏染色和TUNEL染色觀察腦組織病理改變和神經(jīng)元凋亡情況;通過伊文氏藍(lán)法和腦組織含水量的測(cè)定觀察腦組織血腦屏障的通透性;通過Elisa或比色法檢測(cè)血漿和海馬組織中TNF-α、IL-1β、HMGB1、IL-10的濃度,SOD和CAT的活性及MDA和8-iso-PGF2α的水平;通過Western Blot檢測(cè)總Nrf2、核Nrf2及HO-1的蛋白表達(dá)情況;通過免疫熒光染色觀察各組小鼠海馬組織中Nrf2的表達(dá)及核轉(zhuǎn)位情況;通過免疫組化染色觀察各組小鼠海馬組織中HO-1的定位和表達(dá)。結(jié)果:2%氫氣明顯提高膿毒癥小鼠7 d內(nèi)的存活率(P0.05)。在適應(yīng)和訓(xùn)練階段,各組小鼠的僵直時(shí)間百分比無明顯差異(P㧐0.05);于假手術(shù)或CLP后3~14 d,氫氣明顯提高膿毒癥小鼠的交替百分比(P0.01);氫氣顯著提高膿毒癥小鼠的僵直時(shí)間百分比(P0.01);在隱蔽站臺(tái)試驗(yàn)中,氫氣明顯縮短膿毒癥小鼠的潛伏期(P0.001),游泳速度無明顯差異(P㧐0.05);在空間探索實(shí)驗(yàn)中,氫氣顯著提高膿毒癥小鼠在目標(biāo)象限的時(shí)間百分比和穿過站臺(tái)次數(shù)(P0.001)。在假手術(shù)或CLP后1 h和6 h氫氣吸入后不同時(shí)間點(diǎn),Sham+H2組和CLP+H2組的動(dòng)脈血、靜脈血和腦組織中氫氣濃度均較CLP組均明顯提高(P0.05),而在停止吸入氫氣后不同時(shí)間點(diǎn),CLP組和CLP+H2組動(dòng)脈血?dú)錃鉂舛鹊陀赟ham+H2組(P0.05)。于假手術(shù)后24 h時(shí),Sham組和Sham+H2組小鼠海馬CA1區(qū)正常椎體神經(jīng)元數(shù)量無明顯差異,基本無神經(jīng)元凋亡。于CLP后24 h時(shí),與Sham組相比,CLP組和CLP+H2組小鼠海馬CA1區(qū)正常椎體神經(jīng)元數(shù)量減少,凋亡神經(jīng)元數(shù)量增多,EB含量和腦組織含水量增多(P0.05);與CLP組相比,CLP+H2組小鼠海馬CA1區(qū)正常椎體神經(jīng)元數(shù)量明顯增多,凋亡神經(jīng)元明顯減少,EB含量和腦組織含水量明顯減少(P0.05)。于假手術(shù)或CLP后24 h時(shí),與Sham組相比,CLP組和CLP+H2組血漿和海馬組織勻漿中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,總Nrf2表達(dá)增多,部分轉(zhuǎn)位至胞核中,HO-1表達(dá)和HO-1陽(yáng)性細(xì)胞數(shù)均增多(P0.05);與CLP組相比,CLP+H2組小鼠血漿和海馬組織勻漿中TNF-α、IL-1β和HMGB1水平均明顯降低,IL-10水平明顯增高,SOD和CAT的活力均明顯提高,MDA和8-iso-PGF2α水平均明顯降低,總Nrf2表達(dá)明顯增多,細(xì)胞核內(nèi)Nrf2明顯增多,HO-1表達(dá)和HO-1陽(yáng)性細(xì)胞數(shù)均顯著增多(P0.05)。Sham組和Sham+H2組各項(xiàng)指標(biāo)無明顯差異(P㧐0.05)。結(jié)論:2%氫氣吸入可明顯提高膿毒癥小鼠的存活率,改善膿毒癥小鼠的短期工作記憶和長(zhǎng)期恐懼記憶能力,還可提高其學(xué)習(xí)和空間識(shí)別記憶能力,同時(shí)還可明顯改善膿毒癥小鼠腦組織病理?yè)p傷,減少神經(jīng)元凋亡及減輕血腦屏障的破壞,其保護(hù)作用可能通過激活Nrf2/HO-1通路,來抑制炎癥反應(yīng),提高抗氧化酶的活性,降低氧化產(chǎn)物的水平,從而減輕腦組織損傷。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R459.7

【共引文獻(xiàn)】

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