硫氢化钠对脓毒症小鼠心肌线粒体的保护作用及机制研究

发布时间：2018-04-06 20:02

本文选题：脓毒症　切入点：硫氢化钠　出处：《南方医科大学》2017年博士论文

【摘要】：目的:选择最合适盲肠结扎部位行盲肠结扎穿刺法(cecal ligation and puncture,CLP),建立本实验最适脓毒症模型,首次探讨脓毒症对小鼠心肌线粒体生物合成的影响;给予不同浓度硫氢化钠(sodium hydrosulfide,NaHS)干预,首次探讨NaHS对脓毒症小鼠心肌线粒体的保护作用、最佳浓度及相关机制,为脓毒症动物实验研究及临床诊治提供理论基础。方法:1.取40只C57BL/6小鼠随机分为假手术组(Sham)和CLP1、CLP2、CLP3组(分别结扎盲肠远端1/4、1/2、3/4),比较小鼠生存情况,选择最佳模型和取材时间点。2.另取50只C57BL/6小鼠随机分为CLP、CLP + NaHS(4亚组,NaHS浓度分别为25、50、100、2000μmol/L)5组,比较小鼠生存情况。3.另取70只C57BL/6小鼠随机分为正常对照组(Control)、Sham、CLP、CLP+NaHS(4亚组,NaHS浓度同前)7组,检测和比较以下指标:(1)小鼠脓毒症表现。(2)血浆内毒素水平(3)心肌损伤:HE染色观察心肌细胞形态、ELISA检测血清肌钙蛋白(cardiac troponin I,cTnI)水平。(4)心肌线粒体损伤:透射电镜观察线粒体形态学,分光光度法检测线粒体肿胀程度,生物发光法检测ATP水平。(5)氧化应激:TBA 法检测丙二醛(malondialdehyde,MDA)水平,DCFH-DA荧光探针法检测活性氧(reactive oxygen species,ROS)水平。(6)抗氧化物:比色定量法检测谷胱甘肽(glutathione,GSH)水平;Westem Blot法检测锰超氧化物歧化酶(Mn-superoxide dismutase,MnSOD)、血红素加氧酶 1(heme oxygenase-1,HO-1)蛋白水平;RT-qPCR 法检测 MnSOD、HO-1 mRNA水平。(7)线粒体生物合成相关因子:RT-qPCR法检测过氧化物酶体增殖物激活受体 γ 辅激活子 1α(peroxisome proliferator-activated receptor-γ coactivator-1α,PGC-1α)、核因子 E2 相关因子 2(nuclear factor-erythroid-2-related factor 2,Nrf2)、线粒体转录因子 A(mitochondrial transcription factor A,Tfam)mRNA 水平。结果:1.CLP1、CLP2、CLP3组小鼠建模后开始死亡时间分别是12h、11h、8h,CLP1组小鼠48h存活率高于CLP2、CLP3组,故选择CLP1为本研究实验模型,建模后12h为取材时间点。2.NaHS干预组小鼠48h存活率高于CLP组,100μmol/L组最高。3.CLP组小鼠建模后6小时出现脓毒症表现,逐渐加重;NaHS干预组脓毒症表现减轻,NaHS浓度越高,症状越轻。4.CLP组内毒素水平升高。5.CLP组可见HE染色心肌细胞形态异常、cTnI水平升高;NaHS干预组心肌细胞形态异常减轻、cTnI水平下降,NaHS浓度越高作用越明显。6.CLP组心肌线粒体形态异常、肿胀程度增加、ATP水平下降;NaHS干预后线粒体形态异常及肿胀程度减轻,ATP水平升高,NaHS浓度越高作用越明显。7.CLP组ROS、MDA水平升高;与CLP组比较,各浓度NaHS组MDA水平均下降,但100、2000μmol/L组间无显著性差异,ROS水平仅100、200μmol/L组下降。8.CLP组GSH水平下降、MnSOD、HO-1蛋白及mRNA水平上升;NaHS干预组其水平均较CLP组上升,且呈NaHS浓度依赖性,200μmol/L组MnSOD、HO-1蛋白水平低于100μmol/L组。9.CLP 组 PGC-1α、Nrf2、Tfam mRNA 水平均上升;NaHS 干预组较 CLP组继续上升,NaHS浓度越高,上升越明显。10.CLP组和NaHS干预组cTnI水平、线粒体肿胀程度均与ROS、MDA水平呈正相关,ATP水平与ROS、MDA水平呈负相关;NaHS干预组ROS、MDA水平与GSH水平、MnSOD及HO-1 mRNA水平呈负相关,ATP、GSH水平及MnSOD、HO-1、Tfam mRNA 水平均与 PGC-1α、Nrf2 mRNA 水平呈正相关。结论:1.脓毒症可造成心肌线粒体氧化应激损伤。2.NaHS呈浓度依赖性减轻脓毒症时心肌线粒体损伤,100μmol/L为最佳浓度。3.NaHS可通过PGC-1α、Nrf2上调脓毒症时抗氧化物水平、促进线粒体生物合成,从而保护心肌线粒体。
[Abstract]:Objective: to choose the most suitable site for cecal ligation and cecal ligation and puncture method (cecal ligation and puncture, CLP), to establish the optimal model of sepsis in this study, for the first time to investigate the effect of sepsis on myocardial mitochondrial biogenesis in mice; with different concentrations of sodium hydrosulfide (sodium hydrosulfide, NaHS) for the first time to investigate the protective effect of NaHS intervention. In sepsis mice myocardial mitochondria, the optimal concentration and related mechanism, provide a theoretical basis for the study of animal experiment and clinical diagnosis and treatment of sepsis. Methods: 1. 40 C57BL/6 mice were randomly divided into sham operation group (Sham) and CLP1 group (CLP3, CLP2, 1/4,1/2,3/4 were ligated distal caecum), mice survival choose the best model, and at each time point.2. the other 50 C57BL/6 mice were randomly divided into CLP, CLP + NaHS (4 sub group, NaHS concentration was 25,501002000 ~ mol/L) 5 groups, mice survival.3. another 70 C5 7BL/6 mice were randomly divided into normal control group (Control), Sham, CLP, CLP+NaHS (4 sub group, the concentration of NaHS was 7) group to examine and compare the following indicators: (1) in mice with sepsis. (2) the level of plasma endotoxin (3) myocardial injury: Observation of myocardial cell morphology by HE staining, detection serum cardiac troponin ELISA (cardiac troponin I, cTnI) level. (4) myocardial mitochondria: To observe mitochondrial morphology by transmission electron microscopy, spectrophotometric detection of mitochondrial swelling, detect the level of ATP bioluminescence method. (5) oxidative stress: detection of C TBA two (malondialdehyde, MDA) level of formaldehyde, detection of reactive oxygen species DCFH-DA fluorescence probe method (reactive oxygen species, ROS) level. (6) the antioxidant glutathione detection: more than color quantitative method (glutathione, GSH); Westem Blot method for detection of manganese superoxide dismutase (Mn-superoxide dismutase, MnSOD), heme oxygenase 1 (heme oxygenase-1, HO-1 ) protein level; detection of MnSOD RT-qPCR, HO-1 mRNA. (7) factors related to mitochondrial biogenesis: detection of peroxisome proliferator activated receptor gamma RT-qPCR coactivator 1 alpha (peroxisome proliferator-activated receptor- coactivator-1 PGC-1 gamma alpha, alpha), nuclear factor E2 related factor 2 (nuclear factor-erythroid-2-related 2 factor, Nrf2), mitochondrial transcription factor A (mitochondrial transcription factor A, Tfam mRNA). Results: 1.CLP1, CLP2, CLP3 group of mice after death started modeling time were 12h, 11h, 8h, CLP1 group of mice survival rate of 48h is higher than CLP2, CLP3 group, the choice of CLP1 as the experimental model, after modeling 12h at each time point.2.NaHS the intervention group mice survival rate of 48h is higher than that of CLP group, sepsis, 6 hours of 100 mol/L were the highest in.3.CLP group after modeling gradually increased; NaHS intervention group sepsis reduction Light, the higher the concentration of NaHS, the more mild symptoms endotoxin level increased in.4.CLP group.5.CLP group showed HE staining abnormal myocardial cell morphology, elevated levels of cTnI; NaHS group of abnormal myocardial cell morphology intervention decreased, cTnI decreased, the higher the concentration of NaHS is more obvious in.6.CLP group myocardial mitochondrial abnormal morphology, swelling degree increased, the level of ATP after the intervention of NaHS decreased; abnormal mitochondrial morphology and reduce swelling, increased the level of ATP, the higher the concentration of NaHS is more obvious in.7.CLP group ROS, MDA level increased; compared with group CLP, the concentration of NaHS group MDA level decreased, but 1002000 mol/L had no significant difference, ROS level is only 100200 ~ mol/L group the level of GSH decreased in.8.CLP group decreased, MnSOD, HO-1 and mRNA protein level increased; the level of NaHS in the intervention group were lower than CLP group increased, and showed NaHS concentration dependent, 200 mol/L MnSOD group, a low level of HO-1 protein in 100 mol/L group.9.CLP group PGC -1 alpha, Nrf2, Tfam and mRNA levels were increased; the NaHS intervention group compared with the CLP Group continues to rise, the higher the concentration of NaHS increased more obviously,.10.CLP group and NaHS group the level of cTnI, the swelling of mitochondria and ROS, MDA level was positively correlated with ATP level and ROS level of MDA was negatively correlated; NaHS intervention group ROS, MDA level and GSH level was negatively correlated with MnSOD and HO-1 mRNA ATP, HO-1 GSH level and MnSOD, Tfam, mRNA and PGC-1 levels were positively related to the level of mRNA alpha, Nrf2. Conclusion: 1. sepsis can cause myocardial mitochondrial oxidative stress damage in.2.NaHS in a concentration dependent manner to reduce damage of myocardial mitochondria in sepsis when 100 mol/L was the best concentration of.3.NaHS by PGC-1 alpha, Nrf2 upregulated the antioxidant levels in sepsis, promote mitochondrial biogenesis, thereby protecting myocardial mitochondria.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R459.7

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