Protective Effects Of Continuous Insulin Infusion On Liver Mitochondrial Injuries In Early Septic Rats

ObjectiveSepsis is the leading cause of death in PICU. It may cause MODS if it isn't treated in time. Mitochondrion which may be injured in the early stage of sepsis, is the main organelle to produce energy. Its injury is associated with the development of MODS in sepsis. So to find an effective way to protect mitochondrion is very important to decrease the morbility of MODS and the mortality of sepsis. Recently, it has been proved that insulin not noly can reduce blood glucose level, but also have the effects of anti-inflammatory, immunological regulation, attenuating oxidative stress, protecting endothelial cell and improving lipid metabolism. Besides, liver is the most important organ to operate substance and energy metabolism in the body and it is easily to be injured in the early stage of sepsis. So in order to find out whether continuous insulin infusion could protect liver mitochondrion in the early stage of sepsis or not and its associated mechanism, we assessed the function, oxidative stress and morphology of liver mitochondrion. We also evaluated the inflammatory reaction, blood glucose and lipid metabolism of the body. Through the research, we try to find a new way to decrease the morbility of MODS and the mortality of sepsis.Methods1. Animal preparationThe septic model was set up by receiving lipopolysaccharide 10mg/kg by intraperitoneal injection. Twenty four SD rats were randomly divided into three groups (every rat was performed with an external jugular vein catheterization a day before intraperitoneal injection): saline control group (n=8), LPS group (n=8) and insulin therapy group (n=8). Saline control group animals received 0.9% saline only. LPS group animals were intraperitoneal injected with lipopolysaccharide 10mg/kg, and then received an infusion of 0.9% saline 1ml/h. Insulin therapy group animals received an infusion of insulin solution 1ml/h after being intraperitoneal injected LPS 10mg/kg. The dose of insulin is 0.25U/kg/h.2. Measurements and methods(1) General state of animals①Rectal temperature, heart rate and breathing rate were observed before intraperitoneal injection and at 2nd hour, 6th hour, 12th hour, 24th hour after intraperitoneal injection.②Blood glucose levels were monitored at the time point mentioned above.(2) Markers of inflammatory reaction①At 2nd hour and 6th hour after intraperitoneal injection, serum TNF-αand IL-6 were assessed by ELISA.②At 24th hour after intraperitoneal injection, serum C reactive protein were assessed by ELISA.(3) Markers of biochemistry①Liver function: Serum ALT and AST were detected by automatic biochemistry analyzer at 24th hour after intraperitoneal injection.②Lipid metabolism: Serum TG, HDL and LDL were detected by automatic biochemistry analyzer at 24th hour after intraperitoneal injection.(4) Markers of liver mitochondrial injury①At 24th hour after intraperitoneal injection, mitochondrion of liver were isolated by differential centrifugation. Swelling and membrane potential of liver mitochondrion in rats were analyzed by FCM.②At 24th hour after intraperitoneal injection, mitochondrial oxidative stress level (SOD and MDA) of liver in rats were detected by chromatometry.③At 24th hour after intraperitoneal injection, liver mitochondrion were observed through electronic microscopy and the degree of injury in liver mitochondrion were analyzed by Flameng semiquantitative evalution of ultrastructure.3. Statistical analysisThe datas were analyzed by SPSS13.0. All results were reported as mean±standard deviation(±s). Differences between groups were determined by ANOVA. If there was statistical significance and homogeneity of variance between groups, Least-significant difference (LSD) was used. If there was statistical significance and heterogeneity of variance, we used Games-Howell. Pearson correlation test was used when analyzing the correlation between membrane potential of mitochondrion and other datas. P values<0.05 was considered significant.Results1. General state of animals All animals in LPS group and insulin therapy group showed septic symptoms and signs.Compared with control group, blood glucose increased significantly in LPS group (P<0.05). In insulin therapy group, blood glucose reduced significantly compared with LPS group (P<0.05).2. Markers of inflammatory reaction(1) Serum TNF-αand IL-6In LPS group, serum TNF-αand IL-6 increased significantly compared with control group(P<0.05). In insulin therapy group, serum TNF-αand IL-6 decreased significantly compared with LPS group(P<0.05).(2) Serum C reactive proteinCompared with control group, serum C reactive protein increased significantly both in LPS group and insulin therapy group (P<0.05). There was no significant difference in C reactive protein between LPS group and insulin therapy group (P>0.05).3. Markers of biochemistry(1) Liver functionSerum ALT and AST in LPS group were significantly higher than that in control group (P<0.05). Compared with LPS group, serum ALT and AST decreased significantly in insulin therapy group (P<0.05).(2) Lipid metabolismThe difference wasn't significant in serum TG, HDL and LDL among three groups (P>0.05).4. Markers of liver mitochondrial injury(1) Swelling and membrane potential of liver mitochondrion The difference wasn't significant in Swelling of liver mitochondrion among three groups (P>0.05). Compared with control group, membrane potential of liver mitochondrion decreased significantly both in LPS group and insulin therapy group (P<0.05). Membrane potential of liver mitochondrion in insulin therapy group were significantly higher than that in LPS group (P<0.05).(2) Oxidative stress level in liver mitochondrionThe activities of SOD in liver mitochondrion both in LPS group and insulin therapy group were significantly higher than that in contral group (P<0.05). Compared with the LPS group, the activities of SOD decreased significantly in insulin therapy group (P<0.05).There was no significant difference in MDA levels of liver mitochondrion among three groups (P>0.05).(3) Morphology of liver mitochondrionThe changes of liver mitochondrial ultrastructure was not obviouse and no significant difference in its semiquantitative evaluation scores among three groups (P>0.05).5. Correlation between membrane potential of liver mitochondrion and other datas Membrane potential of liver mitochondrion was inversely correlated with serum ALT and AST levels(r=-0.869,P<0.05;r=-0.846,P<0.05).There was no significant correlated relationship between membrane potential of liver mitochondrion and serum TG, HDL and LDL level(sr=-0.309,P>0.05;r=0.209,P>0.05;r=0.264,P>0.05). Membrane potential of liver mitochondrion was inversely correlated with serum TNF-αand IL-6 levels at 6h after intraperitoneal injection(r=-0.879,P<0.05;r=-0.900,P<0.05).Membrane potential of liver mitochondrion was inversely correlated with blood glucose at 6h and at 12h after intraperitoneal injection(r=-0.696,P<0.05;r=-0.744,P<0.05).Conclusions1. There are obviouse injuries of liver function in the early stage of septic rats. The injuries of liver function may have relationship with the injuries of liver mitochondrion. 2. In the early stage of septic rats, functional injuries of liver mitochondrion are obviouse, but the damage of liver mitochondrial ultramicrostructure does not exist obviously. So we can learn that liver mitochondrial injury in the early stage of septic rats is reversible. So the key point to protect liver mitochondrion in septic rats is to treat in its early stage.3. Continuous insulin infusion can protect liver mitochondrion in early stage of septic rats. This effect may work by attenuating inflammatory reaction, decreasing oxidative stress level and blood glucose level. And this effect may have no relationship with lipid metabolism in the early stage of septic rats.