动物实验,脓毒性脑病 z-bo 2020-08-25 478

　　Septic encephalopathy (Sepsis-related encephalopathy, SAE) is manifested as long-term autonomic dysfunction, irritation and cognitive dysfunction, but the exact mechanism is still unclear, and there is no effective treatment. Studies have shown that mitochondrial dysfunction plays an important role in the occurrence and development of multiple organ failure in sepsis. It is speculated that mitochondrial dysfunction is one of the pathophysiological mechanisms of SAE. Mitochondrial antioxidant peptide SS31 can remove mitochondrial reactive oxygen species (ROS) and increase the level of 5'adenosine triphosphate (ATP) through obvious mitochondrial protection. This study observed the effects of SS31 on the cognition and mitochondrial function of SAE mice, and provided reference materials for the prevention and treatment of SAE. Materials and methods Animal selection and grouping were provided by Nanjing General Hospital of Nanjing Military Region, weighing 20-28 g, and forming 60-60 male C57BL/6 mice each year. Randomly divided into 4 groups: sham operation + normal saline group (SN group, n10), sham operation + SS-31 group (ss group, Zhouyilo), CLP + normal saline group (CN group, n20) and CLP + SS31 group ( CS group, n20).

　　CN and CS groups used cecal ligation and perforation (CLP) to establish SAE models, while SN and SS groups only underwent laparotomy, separation of the distal cecum and abdominal closure. After the operation, 5 mg/kg of SS- (31 (SS and CS group, Shanghai Qiangyao Biotechnology Co., Ltd.) and equal volume of saline (SN and CN group) were injected intraperitoneally for 6 consecutive days. Establishment and fasting before the operation 12 In 1 hour, pentobarbital sodium (40 mg/kg, sigma) was injected intraperitoneally for anesthesia, and a 1 cm incision was made between the midline of the abdomen and the intestine. 1.2 The CI11 of the proximal root of the cecum was 4-0, and it was looped with silk thread Ligation, puncture the end of the ligation with a 22-gauge needle twice to squeeze the urine and place the parts and suture the peritoneum and skin in sequence, and complete all operations within 8 minutes. Behavioral test According to the literature, the mice that survived 7 days after surgery Perform a conditioned fear experiment. Place the mouse in an experiment box wiped with 75% alcohol for 180 seconds, and then perform 60 S sound stimulation (80 dB, 3600 Hz), and then perform electrical stimulation on the foot (0.75 mA, 1 second) ), the mouse adapts to 180 S. 2 and 24 hours after training, live and acoustic experiments were carried out. The live experiments mainly reflected the hippocampus-dependent memory, while the acoustic experiments mainly focused on the hippocampus. Reflecting independent memory V, E8I. Experiments in a scene In, put the mouse on the same ring for 390S, and record the freezing time in the second 180S; in the acoustic experiment, place the mouse in a different environment.

　　During the experiment, the same sound stimulus was applied for 180 seconds without electric shock, and its duration was recorded. During the experiment, the SuperFCS system (Shanghai Xinruan Company) was used to automatically determine the mouse’s position in the shock box. Active status. After each experiment, scrub the substrate with 75% alcohol to avoid disturbing the smell. After ROS and ATP level detection behavior test, 5 mice in each group were sacrificed and fresh. Instructions for the detection of ROS and ATP levels by various ROS kits (Haijiemei Pharmaceutical Technology Co., Ltd.) and ATP kits (Haibi Yuntian Biotechnology Co., Ltd.) of various hippocampal tissue homogenization technologies. The enzyme activity and mitochondrial membrane potential (MMP) detection behavior of complexes I, II, III, IV were tested by killing 5 mice in each group to obtain fresh hippocampal tissue. Please refer to the mitochondrial extraction kit (Shanghai Biyuntian Biotechnology Co., Ltd.). The company's instructions are to extract and purify mitochondria. Follow the instructions of the mitochondrial respiratory chain complex activity detection kit and purified mitochondrial membrane potential fluorescence determination kit (Shanghai Liming Gene Medical Technology Co., Ltd.) to operate the enzyme activity and MMP levels of complexes II, III, and IV.

　　SPSS17 is used for statistical analysis. o Statistical analysis software. The measured data is expressed as the mean±standard deviation (i±s), the comparison between groups uses one-way analysis of variance, and the pairwise comparison uses the SNK method.

　　Result

　　Conditional fear experiment In this study, there were 9 deaths and 6 deaths in the CN and CS groups respectively, and the difference was not statistically significant. The SN and SS groups did not die, but the mice were still alive and completed the conditioned fear experiment. Compared with the CN group, the freezing time of the 24-hour scene experiment in the SN, SS and CS groups increased significantly (P\u003c0.05). There was no statistically significant difference between the two groups of 2-hour scene experiments, acoustic experiments and 24-hour acoustic experiments. versus

　　Compared with the CN group, SN, SS, and CS group, the changes in ROS, ATP, and MMP levels were significantly reduced (P\u003c0.05), and the levels of ATP and MMP were significantly increased (P\u003c0.05)). Compared with the SN and SS groups, ROS levels were significantly reduced, while ATP and MMP levels were significantly increased (P \u003cⅡ,Ⅲ,Ⅳ enzyme activity). Compared with the CN group, SN, SS and CS group compounds, Physics and III activities were significantly increased (P\u003c0.05). Compared with the CS group, the complex I and III activities of the SN and SS groups increased significantly (P\u003c0.05).

　　in conclusion

　　In this study, CLP mice showed a significant decrease in the stiffness of the 24-hour scene on the 8th day after surgery, manifested as hippocampal-dependent cognitive impairment, and increased ATP as the level of hippocampal ROS increased. This level was observed to decrease. The etiology of mitochondrial and SAE cognitive dysfunction in sepsis is the increased ROS level and the decreased ATP level caused by the impaired mitochondrial electron transport chain function, as well as the decreased mitochondrial complex, III enzyme activity and MMP level. Guess one of them. In sepsis, the damage to the mitochondrial electron transport chain of different tissues is different. Comim and his colleagues significantly reduced the mitochondrial complex enzyme activities in the cerebellum, hippocampus, striatum and cerebral cortex of CLP rats at 24, 48, and 96 hours, and significantly changed the enzyme activities of complexes II, III, and IV. . Although not, it was observed that the activity of Davira CLP rat brain complex IV decreased significantly after 24 hours. In addition, Perucci et al. We observed that E113 reduced the enzyme activity of complex II and III in CLP rat skeletal muscle tissue at 12 hours, and reduced the enzyme activity of all complexes at 48 hours. Zapelini et al. observed that the activity of complex IV in liver tissue decreased after 3 to 48 hours. In this study, we observed that the enzyme activities of complexes II and IV in the hippocampus of CLP mice were significantly reduced on the 8th day, and the enzyme activities of complexes II and IV did not change significantly. This change may be related to the CLP timing. The organization is different. Mitochondrial electron transport chain complex and MMP enzyme activity are important parameters of mitochondrial function. Among them, complex and III are the main sites for ROS generation in cells. A decrease in its activity can lead to a significant increase in ROS. MMP is a prerequisite for the formation of ATP. In the process of electron transport, protons enter the mitochondrial membrane through the penetrating complex and the reverse concentration gradient to form linear MMP, and enter the mitochondria through the concentration gradient of ATP synthase to produce ATP. Therefore, the decrease in the activity of the complex and III enzyme leads to a decrease in MMP and therefore a decrease in ATP production.

　　Therefore, in this study, CLP caused mitochondrial dysfunction in the hippocampus of mice, which was mainly manifested as a decrease in mitochondrial complex and III enzyme activity and MMP content, accompanied by an increase in ROS levels and a decrease in ATP levels. Mitochondrial antioxidant peptide SS-31 can freely cross the blood-brain barrier and cell membranes. The inner mitochondrial membrane concentration is 1000 times higher than the outer mitochondrial concentration, and it has nothing to do with the mitochondrial membrane potential. SS31 can remove various intracellular ROS, such as hydrogen peroxide (H.O.), superoxide anion (OF) and hydroxyl (ΦOH). It has the function of increasing ATP production and stabilizing the mitochondrial membrane potential. It plays an important protective role in degenerative diseases, metabolic diseases and ischemia-reperfusion injury. In this study, continuous administration of SS-31 to the SAE mouse model can increase the rigidity of the 24-hour scenario experiment, improve the cognitive function of the mice, and reduce the level of hippocampal ROS and the levels of ATP and MMP. It was found to rise, and complicated work was suppressed. The reduction of III enzyme activity obviously protects mitochondria. All in all, mitochondrial dysfunction may be one of the pathophysiological mechanisms of SAE cognitive dysfunction. Continuous administration of mitochondrial antioxidant peptide SS31 can improve the cognitive function of septic encephalopathy mice, and its effect may be related to the protection of mitochondrial function.