| Background:Sepsis is a common disease in pediatric intensive care unit, which its mortality rate is extremely high. The exact definition of sepsis began in1991American Thoracic Society and the society of critical care medicine joint meeting to reach consensus:sepsis can be defined as the infection caused by systemic inflammatory response syndrome. Now that sepsis is not simply the host immune response to infection, but an inflammation of damaged and non-repaired that can lead to multiple organ dysfunction. Septic cardiomyopathy is a common complication of sepsis, can be represented as a biventricular reversible dilatation, ejection fraction decreased, falling response on the fluid resuscitation and positive inotropic drug. Simultaneously serum troponin, BNP and other cardiac markers rise. Septic cardiomyopathy can increase patients’ mortality; therefore its pathogenesis is attracting more and more attention.The pathogenesis of septic cardiomyopathy is very complex; one of importance mechanism is mitochondrial dysfunction. There has been a clinical finding which the severity of sepsis is related to the damage of mitochondria and energy generation disorder. Mitochondria ultrastructure of myocardial tissue accompanied by mitochondrial respiratory dysfunction has found in sepsis model induced by endotoxin. There are many pathways mechanisms involved in cardiac mitochondrial dysfunction in sepsis. On the one hand the damaged mitochondria leads to a decrease in tissue oxygen utilization ability, called ’cell hypoxia’, at the same time as the structure and function of mitochondria damaged, leading to mitochondrial metabolic changes, therefore the production of mitochondrial ROS increased which can aggravate intracellular oxidative stress. Recently found that mitochondrion may participate in sepsis associated inflammation:Reactive oxygen species can crosstalk with inflammatory pathway oxidative stress can further activate inflammatory reaction; in addition the damaged mitochondria can release cycle of mitochondrial DNA fragments have been determined as mitochondria derived danger associated molecular patterns (DAMPS), can activate inflammation reaction through pattern recognition receptors. Currently mitochondrial dysfunction and molecular pathways of mitochondrion in septic cardiomyopathy needs further understanding, this will help reveal the mechanisms and treatment of septic cardiomyopathy.Mitochondrial uncoupling protein2is a proton transporter is present in the inner mitochondrial membrane; UCP2is widely distributed (including the spleen, thymus, pancreas, heart, lung, white adipose tissue, brain, liver, bone and skeletal muscle, etc.). Now that reported UCP2can regulation of energy ATP generation, inhibition of mitochondrial reactive oxygen species (ROS, Reactive oxygen species), to maintain calcium balance within mitochondria and mitochondrial membrane potential, participate in regeneration of mitochondria and so on. But there have been no reports of its specific role in the mechanism of septic cardiomyopathy. PurposeOn whole animal level, we used intraperitoneal injection of endotoxin established rat model of sepsis, the animals were sacrificed by different time point.Observed the dynamic expression of UCP2in myocardial tissue for further research its specific role in septic cardiomyopathy. At the cellular level, we used septic rats serum stimulated H9C2myocardial cell established vitro model of septic myocardial injury. Through siRNA interference technology, preliminary exploration of the role of UCP2gene in inflammatory pathways and oxidative stress in septic cardiomyopathy cellular model. Our study include two parts, the first part is to research the dynamic expression of UCP2in myocardial tissue of septic rats in mRNA and protein level; the second part is to investigate P-P38, NF-KB expression and oxidative stress of UCP2gene siRNA transfection H9C2cardiomyocytes challenge with septic-serum,Research MethodsPart I:40Healthy adult specific pathogen free (SPF) male SD rats (provided by the Guangdong Medical Laboratory Animal Center), experimental animals were randomly divided into two groups:normal control group (n=8), sepsis group (n=32). Sepsis group divided into (6h,12h,24h,48h) four subgroups. Before the experiment rats were fasted for12h, free access to water. Normal control group without any treatment. Sepsis group with lipopolysaccharide (LPS)10mg/kg intraperitoneal injection. Septic animals were sacrificed at different time points. Specimen Collection:Rat hearts were removed, rinsing with physiological saline, part of the left ventricle portion was immersed in4%paraformaldehyde; rest quickly stored in liquid nitrogen to prepare for RNA and protein extraction.Part II:20Healthy adult specific pathogen free (SPF) male SD rats (provided by the Guangdong Medical Laboratory Animal Center), experimental animals were randomly divided into two groups:normal control group (n=10), sepsis group (n=10). Normal control group without any treatment. Sepsis group with lipopolysaccharide (LPS)10mg/kg intraperitoneal injection. After6h blood obtained by heart puncture,4℃for30minutes, centrifuged at800rpm per minute, the supernatant was collected a biological safety cabinet.-80℃C to save for later use. Cardiomyocytes (H9C2), were randomly divided into control group, normal rat serum,10%serum stimulation in septic rats12h group, UCP2-siRNA interference+10%serum stimulation in rats with sepsis12h group, negative siRNA transfection+10%serum stimulation in septic rats12h group. NO levels in cell supernatants and intracellular levels of MDA and the fluorescent probe to detect ROS levels; cell p-p38MAPK expression and nuclear transcription factor (NF-κB) expression detected by western-bolt.ResultsThe first partDynamic septic myocardial tissue UCP2expression:The statistical test between the groups UCP2mRNA and UCP2protein expression were significantly different (UCP2mRNA:F value=73.96, P<0.01; UCP2protein:F value=101.82, P <0.01).The relative expression levels of UCP2mRNA in each sepsis group higher than normal group (P<0.01), with the highest level of UCP2mRNA expression at sepsis24h group; UCP2protein gray value comparison shows that sepsis increased UCP2protein relative expression level of each group,6h,12h,24h,48h group than the control group high (P<0.01), and mRNA expression similar trend, with the highest expression levels at24h. Upright microscope showed immunohistochemistry results that a small amount of UCP2protein expression in normal rat myocardium, localized in the cytoplasm. Lighter yellow stained in the control group, deepest dye stained sepsis24h group.1Expression of p-p38MAPK, NF-κB in H9C2myocardial cells in each group was detected by Western blot. Statistical analysis significant differences among the groups (p-p38:F=118.40, P<0.01; NF-B:F-156.98, P<0.01). The expression of p-p38MAPK and NF-κB in normal rat serum group were no significant difference compared to normal control group (P>0.05). The expression of p-p38MAPK and NF-κB in10%septic rats serum12h group were significant increased compared with normal control group (P<0.01); The expression of p-p38MAPK and NF-κB in UCP2-siRNA interference+10%septic rats serum stimulated12h group were significantly increased compared with10%septic rats serum12h group (P<0.01); The expression of p-p38MAPK and NF-κ B in negative transfection of siRNA+10%10%septic rats serum stimulated12h group were no significant difference compared to10%septic rats serum12h group (P>0.05).Detection of the expression level of NO cell supernatantThe expression level of NO between groups by statistics difference (F=65.01, P<0.01). Compared with the normal control group, the expression level of NO in cell supernatant of10%septic rats serum12h group was increased significantly (P<0.01); the expression level of NO in cell supernatant of10%septic rats serum12h group no significant difference compare with UCP2-siRNA interference+10%septic rats serum stimulated12h group (P>0.05)3> Detection of MDA level in cell lysates.Among the expression level of MDA cell lysate by statistics significant difference (F=66.37, P<0.01). Compared with the normal control group, the expression level of MDA in cell lysates.of10%septic rats serum12h group was increased significantly (P<0.01); the expression level of MDA in cell lysates10%UCP2-siRNA interference+10%septic rats serum stimulated12h group was significant increased compare with septic rats serum12h group (P>0.05)4^Fluorescence intensity of cells were observed by fluorescence microscopeWith green fluorescence appeared in each groups of cells, green fluorescence distributed uniformly in the cells. Normal control group and normal rat serum group intracellular fluorescence was weak, the septic serum stimulation group cells with fluorescence were higher than normal group increased to some degree, the green fluorescence intensity is the strongest in10%UCP2-siRNA interference+10%septic rats serum stimulated12h group, suggesting the highest level of reactive oxygen species.Conclusion1In the experiment model in septic rats, confirmed myocardial tissue UCP2expression increased.2Through the establishment of septic myocardial injury vitro model, interference of UCP2expression can further promote the activation of P-P38and NF-KB expression, and intracellular oxidative stress3UCP2is involved in the pathogenesis of septic cardiomyopathy, may play a role by oxidative stress and inflammatory pathway. |
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