水通道蛋白8对脓毒症大鼠肝细胞线粒体形态的影响
发布时间：2018-09-12 08:59
【摘要】：脓毒症(sepsis)是指感染引起的全身性炎症反应综合征(systemic infla-mmatory response syndrome, SIRS),是危重症患者病死的主要原因之一,进一步发展可引起重症脓毒症(severe sepsis)脓毒症休克(septic shock)和多器官功能障碍综合征(multiple organ dysfunc-tion syndrome, MODS)肝脏是脓毒症状态下最易受累的器官之一,肝功能不全是脓毒症发展为多器官功能衰竭(multiple organ failure,MOF)的早期表现之一脓毒症的发病机制尚不十分明确,可能的发病机制包括:炎症因子风暴凝血系统紊乱和微循环障碍免疫抑制、线粒体功能障碍和机体氧供与氧耗失衡等肝细胞线粒体是机体营养物质和能量代谢中心,线粒体功能障碍在脓毒症状态下,尤其是脓毒症休克患者的病情发展中起着很重要的作用水通道蛋白8(aquaporin-8,AQP8)是在肝细胞线粒体内膜(inner mitochondria membrane,IMM)上表达的一种水分子通道,是具有高度选择性和高效转运水分子的特异孔道,对维持肝细胞线粒体的结构和功能具有较大的影响本研究使用盲肠结扎穿孔法制备脓毒症大鼠模型,旨在探讨脓毒症造成肝细胞线粒体形态的变化,以及线粒体内膜AQP8在脓毒症介导的肝损伤中的所发挥的作用 目的 研究脓毒症状态下水通道蛋白8(AQP8)在肝细胞线粒体膜上表达程度对线粒体形态变化的影响,为进一步探寻改善线粒体膜AQP8蛋白表达的方法,以期能起到对脓毒症介导肝损伤的保护作用 方法 24只SD大鼠随机分为2组:对照组和脓毒症组,每组12只采用盲肠结扎穿孔术制备脓毒症实验动物模型,对照组进行假手术,完成除盲肠结扎穿孔外的实验过程观察两组动物的体温呼吸频率心率变化和对外界刺激的反应,达到脓毒症诊断标准后继续观察1h,然后在麻醉后用断头法处死实验大鼠;取出大鼠肝脏,一部分新鲜肝脏用于制作电镜标本,进行超微结构观察,另一部分肝脏用于提取线粒体,后采用Western-blotting法测定线粒体AQP8蛋白表达水平,并用RT-PCR法检测肝细胞线粒体AQP8mRNA水平采用SPSS13.0统计软件进行分析计量资料采用单因素方差分析(one-wayANOVA) 结果 1.线粒体超微结构变化 对照组肝细胞膜边界清楚完整,内质网及线粒体数目较多且形态正常而脓毒症组肝细胞线粒体显著肿胀嵴变宽短,并出现线粒体膜破裂,内质网也出现扩张断裂及空泡化 2.肝细胞线粒体AQP8蛋白表达 以prohibitin作为内参,用AQP8/prohibitin表示AQP8蛋白相对表达水平,将对照组的AQP8蛋白相对表达水平设为1,计算出脓毒症组(n=12)AQP8蛋白相对表达水平为0.61±0.08,较对照组减少平均约39%,差异有显著统计学意义(F=62.71,P0.01) 3肝细胞线粒体AQP8mRNA的表达 以β-actin作为内参,AQP8/β-actin表示AQP8mRNA相对表达量,将对照组的AQP8mRNA相对表达水平设为1,脓毒症组AQP8mRNA相对表达量为1.59±0.24;AQP8mRNA表达较对照组增加平均约59%,差异有显著统计学意义(F=67.85, P0.01) 结论 1.采用盲肠结扎穿孔术可以成功制备大鼠脓毒症模型; 2.脓毒症可以介导急性肝损伤,肝细胞线粒体超微结构发生明显的变化; 3.脓毒症状态下肝细胞线粒体AQP8蛋白表达下调,可能是导致线粒体肿胀变性的重要因素之一; 4.脓毒症状态下肝细胞线粒体AQP8蛋白表达下调,而AQP8mRNA表达增加,提示可能存在代偿机制,但由于存在转录后抑制AQP8蛋白并未能回升
[Abstract]:Sepsis is a systemic inflammation-mmatory response syndrome (SIRS) caused by infection, which is one of the main causes of death in critically ill patients. Further development of sepsis shock and multiple organ dysfunction syndrome (MODS) can cause severe sepsis. Unc-tion syndrome (MODS) liver is one of the most susceptible organs in sepsis. Liver dysfunction is one of the early manifestations of sepsis developing into multiple organ failure (MOF). The pathogenesis of sepsis is still unclear. Possible pathogenesis includes: disturbance of the inflammatory factor storm coagulation system and microorganisms. Hepatocyte mitochondria, such as circulatory dysfunction, immune suppression, mitochondrial dysfunction, and imbalance of oxygen supply and consumption, are the center of nutrients and energy metabolism. Mitochondrial dysfunction plays an important role in the development of septic symptoms, especially in patients with septic shock. A water molecule channel expressed on the inner mitochondria membrane (IMM) is a highly selective and efficient channel for transporting water molecules. It has a great influence on the maintenance of the structure and function of hepatocyte mitochondria. In this study, we used cecum ligation and perforation method to prepare sepsis rat model. Morphological changes of hepatocyte mitochondria and the role of mitochondrial endometrial AQP8 in sepsis-mediated liver injury
objective
To study the effect of aquaporin 8 (AQP8) expression on mitochondrial morphology of hepatocytes in septic symptoms, and to explore a method to improve the expression of AQP8 protein on mitochondrial membrane, so as to protect liver from sepsis-induced liver injury.
Method
Twenty-four SD rats were randomly divided into two groups: control group and sepsis group. Twelve rats in each group were subjected to cecum ligation and perforation to establish sepsis experimental animal model. The control group was subjected to sham operation. The experimental process except cecum ligation and perforation was completed. The changes of body temperature, respiratory rate, heart rate and response to external stimuli were observed in the two groups to achieve sepsis diagnosis. After 1 hour, the rats were killed by decapitation after anesthesia. The liver of the rats was taken out, some of the fresh liver was used for making electron microscopic specimens, and the other part of the liver was used for extracting mitochondria. The expression of AQP8 protein in mitochondria was determined by Western blotting, and the hepatocytes were detected by RT-PCR. Mitochondrial AQP8 mRNA levels were analyzed by SPSS13.0 statistical software and measured by one-way ANOVA.
Result
1. mitochondrial ultrastructural changes
In the control group, the boundary of hepatocyte membrane was clear and intact, the number of endoplasmic reticulum and mitochondria was large and the morphology was normal, but in the sepsis group, the mitochondrial swelling cristae became wider and shorter, and the mitochondrial membrane ruptured, the endoplasmic reticulum distended to rupt and vacuole.
2. expression of mitochondrial AQP8 protein in hepatocytes
Using Prohibitin as internal reference, AQP8/prohibitin was used to express the relative expression level of AQP8 protein. The relative expression level of AQP8 protein in control group was set to 1. The relative expression level of AQP8 protein in sepsis group (n=12) was calculated to be 0.61 (+ 0.08), which was about 39% lower than that in control group (F=62.71, P 0.01).
3 expression of mitochondrial AQP8mRNA in hepatocytes
Using beta-actin as internal reference, AQP8/beta-actin expressed the relative expression level of AQP8 mRNA. The relative expression level of AQP8 mRNA in control group was set to 1, AQP8 mRNA in sepsis group was 1.59 [0.24] and AQP8 mRNA expression was increased by 59% on average compared with control group (F = 67.85, P 0.01).
conclusion
1. the sepsis model can be successfully prepared by cecal ligation and puncture.
2. sepsis can induce acute liver injury, and the ultrastructure of mitochondria of liver cells is obviously changed.
3. The down-regulation of AQP8 protein expression in hepatocyte mitochondria under septic symptoms may be one of the important factors leading to mitochondrial swelling and degeneration.
4. The expression of AQP8 protein in hepatocyte mitochondria was down-regulated and the expression of AQP8 mRNA was up-regulated in septic symptoms, suggesting that there may be compensatory mechanism, but the inhibition of AQP8 protein after transcription did not recover.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7
[Abstract]:Sepsis is a systemic inflammation-mmatory response syndrome (SIRS) caused by infection, which is one of the main causes of death in critically ill patients. Further development of sepsis shock and multiple organ dysfunction syndrome (MODS) can cause severe sepsis. Unc-tion syndrome (MODS) liver is one of the most susceptible organs in sepsis. Liver dysfunction is one of the early manifestations of sepsis developing into multiple organ failure (MOF). The pathogenesis of sepsis is still unclear. Possible pathogenesis includes: disturbance of the inflammatory factor storm coagulation system and microorganisms. Hepatocyte mitochondria, such as circulatory dysfunction, immune suppression, mitochondrial dysfunction, and imbalance of oxygen supply and consumption, are the center of nutrients and energy metabolism. Mitochondrial dysfunction plays an important role in the development of septic symptoms, especially in patients with septic shock. A water molecule channel expressed on the inner mitochondria membrane (IMM) is a highly selective and efficient channel for transporting water molecules. It has a great influence on the maintenance of the structure and function of hepatocyte mitochondria. In this study, we used cecum ligation and perforation method to prepare sepsis rat model. Morphological changes of hepatocyte mitochondria and the role of mitochondrial endometrial AQP8 in sepsis-mediated liver injury
objective
To study the effect of aquaporin 8 (AQP8) expression on mitochondrial morphology of hepatocytes in septic symptoms, and to explore a method to improve the expression of AQP8 protein on mitochondrial membrane, so as to protect liver from sepsis-induced liver injury.
Method
Twenty-four SD rats were randomly divided into two groups: control group and sepsis group. Twelve rats in each group were subjected to cecum ligation and perforation to establish sepsis experimental animal model. The control group was subjected to sham operation. The experimental process except cecum ligation and perforation was completed. The changes of body temperature, respiratory rate, heart rate and response to external stimuli were observed in the two groups to achieve sepsis diagnosis. After 1 hour, the rats were killed by decapitation after anesthesia. The liver of the rats was taken out, some of the fresh liver was used for making electron microscopic specimens, and the other part of the liver was used for extracting mitochondria. The expression of AQP8 protein in mitochondria was determined by Western blotting, and the hepatocytes were detected by RT-PCR. Mitochondrial AQP8 mRNA levels were analyzed by SPSS13.0 statistical software and measured by one-way ANOVA.
Result
1. mitochondrial ultrastructural changes
In the control group, the boundary of hepatocyte membrane was clear and intact, the number of endoplasmic reticulum and mitochondria was large and the morphology was normal, but in the sepsis group, the mitochondrial swelling cristae became wider and shorter, and the mitochondrial membrane ruptured, the endoplasmic reticulum distended to rupt and vacuole.
2. expression of mitochondrial AQP8 protein in hepatocytes
Using Prohibitin as internal reference, AQP8/prohibitin was used to express the relative expression level of AQP8 protein. The relative expression level of AQP8 protein in control group was set to 1. The relative expression level of AQP8 protein in sepsis group (n=12) was calculated to be 0.61 (+ 0.08), which was about 39% lower than that in control group (F=62.71, P 0.01).
3 expression of mitochondrial AQP8mRNA in hepatocytes
Using beta-actin as internal reference, AQP8/beta-actin expressed the relative expression level of AQP8 mRNA. The relative expression level of AQP8 mRNA in control group was set to 1, AQP8 mRNA in sepsis group was 1.59 [0.24] and AQP8 mRNA expression was increased by 59% on average compared with control group (F = 67.85, P 0.01).
conclusion
1. the sepsis model can be successfully prepared by cecal ligation and puncture.
2. sepsis can induce acute liver injury, and the ultrastructure of mitochondria of liver cells is obviously changed.
3. The down-regulation of AQP8 protein expression in hepatocyte mitochondria under septic symptoms may be one of the important factors leading to mitochondrial swelling and degeneration.
4. The expression of AQP8 protein in hepatocyte mitochondria was down-regulated and the expression of AQP8 mRNA was up-regulated in septic symptoms, suggesting that there may be compensatory mechanism, but the inhibition of AQP8 protein after transcription did not recover.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R459.7
【共引文献】
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