Research Of Mitochondrial Injury And Mutiple Organ Dysfunction In Early Stage Of Sepsis In Rats

Sepsis is the leading cause of death in ICU,the mechanism of which is complicated and remain to be elucidated.Delayed treatment of sepsis may lead to MODS and MMDS,even death.Early treatment of sepsis could reduce the mortality of sepsis and MODS.In early stage of sepsis,injury of mitochondrion which produce main energy in cell,affect the prognosis of sepsis.In order to find the method of prevent mitochondrion injury,we selected the renal,adrenal,brain and liver of early septic rats as samples to research mitochondrion injury and it's mechanism.1.The animal model:SD rats were devided into several groups randomly according to the detected time.The rats in LPS groups which received 10mg/kg lipopolysaccharide by intraperitoneal injection were positive LPS in blood and got increased serum C reactive protein.2.The design of the research:(1)To study the correlation between mitochondrial injury and organ dysfunction in early sepsis.Renal is extremely vulnerable to sepsis-induced organ failure.Renal dysfunction could influence the function of other organs directly or indirectly,and renal dysfunction could finally lead to MODS.There are plenty of ATP-dependent ion-channels on renal cells,so renal mitochondrial function is directly bound up with renal function.Adrenal is an very important gliand in system stress reaction and can modulate system endocrine.Adrenal dysfunction has significant correlation with the progress of sepsis.Furthermore,adrenal can protect the function of other organs during sepsis.Compared with renal,there might be obvious difference in the capacity of resisting sepsis-induced damage.We observed renal and adrenal mitochondrial ultrestructure of early septic rats,and gave the mitochondrial injury evaluation scores.We also test the ratio of FSC/SSC and mitochondrial membrane potential.We did all these things for the purpose to score renal and adrenal mitochondrial injury.We evaluated renal and adrenal function through levels of serum BUN,Cr and hydrocortison.We studied the correlation between mitochondrial injury and organ function of renal and adrenal.We tried to compare the pathogenesis of renal and adrenal mitochondrial injury through comparing the intensity of oxidative stress in each kind of mitochondria.(2)Experimental research of brain mitochondrial injury in early septic rats. Sepsis-induced brain injury usually occurred in the first 3 hours of onset of sepsis.Brain is an improtant part of CNS,which imposes about 20%-25%of total body energy.Mitochondria are the predominant place of oxidative phosphorylation in cells,so mitochondria function is extremely important for brain function.We tested the level of inflammatory mediators in serum and brain,and some items of oxidative stress,also the correlation among these projects.To study the pathogenesis of brain mitochondrial injury.Try to give an clue for clinical treatment of sepsis-induced early mitochondrial injury.(3)Protective test of liver mitochondrion in the early sepsis.Liver is the main organ for material and energy metabolism,which is rich in mitochondria.The proper function of liver is the essential condition of body metabolism.Insulin which is very important for regulating body metabolism can relieve inflammatory reaction,hypermetabolism and hyperdynamic condition in early sepsis.We compared the rats liver function,liver mitochondrial injury,blood glucose and the intensity of inflammatory reaction of continue insulin infusion group with non-intervention septic rats group.We observed the influence of continue insulin infusion on liver mitochondria and tried to describe the possible pathogenesis.We also tried to find out an possible way to block early sepsis-induced mitochondrial injury.3.The levels in the research:(1)Mitochondrion of each organ was observed by electronic microscopy and the degree of injury was analyzed by Flameng semiquantitative evalution of ultrastructure. The membrane potential and density of mitochondrion was detected by flow cytometry.Activitie of Na~+-K~+ATPase of mitochondrion was also detected.(2)Oxidative stress levels in mitochondrial(activities of SOD,NOS and levels of MDA,NO,GSH) in rats were assessed.Inflammatory factors in blood serum and brain including TNF-α,IL-1βand IL-6 were assessed by ELISA.(3)Injury of structure and function of the organs were analyzed by histologic examination and biochemistry levels in blood serum,such as urea nitrogen and creatinine for renal,Cortisol for adrenal and aminotransferase for liver.The research was composed of three parts:Chapters 3:The correlation between mitochondrial injury and organ function of renal and adrenal in early sepsis.We chose renal and adrenal of early septic rats as subjects to stand for the more susceptible organ and more protectable organ respectively during sepsis.Thirty SD rats were randomly divided into three groups: control group,6-hour LPS group and 24-hour LPS group.T o compare the organ function and mitochondrial status of renal and adrenal between these three groups, and try to describe the pathogenesis.Results:①Renal function and renal mitochondrial were injuried in early septic rats.Compared with control group,the levels of Urea and Cr in LPS groups increased significantly;semiquantitative evaluation scores of renal mitochondria in LPS groups were significantly higher, meanwhile the ratio of FSC/SSC and mitochondria membrane potential of renal mitochondria were significantly lower;there was correlation between renal mitochondrial injury and renal dysfunction in early septic rats.②There was no distinct injury of adrenal function during early sepsis.With the progress of sepsis,the adrenal/body coefficient and the levels of corticosterone in serum of LPS groups were significantly difference as compared with control group.Semiquantitative evaluation scores of adrenal mitochondria in LPS groups were only increased mildly as compared with control groups,and there was no statistic significance.Also compared with control group,the ration of FSC/SSC in adrenal mitochondria were higher and the adrenal mitochondrial membrane potential was lower,but all these changes were reversible.Further,there was no obvious oxidative stress injury in adrenal mitochondria.These results mentioned above indicated that there was renal mitochondria injury in early sepsis,and renal mitochondrial injury was an important causation of renal dysfunction.Oxidative stress was a probable reason for renal mitochondria injury.Adrenal injury was also occurred in early sepsis,but it was not serious.There was no significant evidence of oxidative stress injury in adrenal mitochondria.The results of adrenal in our experiment indicated that adrenal as an participant and protector during system stress response,its mitochondria and organ function were more resistance and more repairable to sepsis induced injury.Chapters 2:Primary study of brain mitochondrial injury in early septic rats. Forty SD rats were randomly divided into control group,3-hour LPS group,6-hour LPS group and 24-hour LPS group to study the brain mitochondrial injury in superacute phase of sepsis and try to explanation the pathogenesis of brain mitochondrial injury.The results showed that 6-hour LPS group rats neuronal and astroglial mitochondrial ultrastructure injury evalution score were increased significantly as compared with control group,but rats microglial and oligodendroglial mitochondrial ultrastructure injury evalution score in all the 4 groups did not have significant difference.Rats brain mitochondrial membrane potential of 3-hour LPS group was decreased significantly as compared with control group,but the brain mitochondrial membrane potential in 6-hour and 24-hour LPS group were not significant difference as compared with control group.The level of brain mitochondrial MDA and NO and the activity of brain mitochondral Mn-SOD and NOS were significant difference between groups.Serum TNF-αof 3-hour and 6-hour LPS groups were significant higher as compared with control group.All these LPS groups had higher serum IL-6 levels as compared with control group.Furthermore, the levels of TNF-α,IL-1βand IL-6 of brain tissue homogenate were increased significantly as compared with control group.Rats serum IL-6 level had negative correlaton with brain mitochondrial membrane potential.Rats brain tissue homogenate level of TNF-αhad positive correaltion with brain mitochondrial Mn-SOD activities,but negative correlation with brain mitochondrial MDA levels. There was obvious negative correlation between septic rats brain tissue homogenate level of IL-1βand barin mitochondrial MDA levels,NOS activities,NO levels.The septic rats brain mitochondrial membrane potential had positive correlation with the activities of brain mitochondrial Mn-SOD,but negative correlation with brain mitochondrial MDA levels,NOS activities and NO levels.Septic rats brain tissue homogenate level of TNF-αhad positive correlation with brain mitochondrial membrane potential,but the level of IL-1βhad negative correlation with brain mitochondrial membrane potential.Rats serum level of IL-6 also had negative correlation with brain mitochondral membrane potential.These results mentioned above indicated that the injury of rats brain mitochondria was reversible in early sepsis and septic rats brain mitochondrial dysfunction occurred more earlier than brain mitochondrial ultrastructure change.Oxidative stress was an important causation of septic rats brain mitochondrial injury.Rats brain mtMn-SOD,mtNOS and mtNO plays crucial roles in brain oxidative stress injury.Inflammatory mediators in septic rats brain,especially TNF-αand IL-1β,gave their different impact on septic rats brain mitochondrial injury:TNF-αhad positive correlation with brain mitochondrial membrane potentil and might play protect role with mitochondrial function;IL-1βhad negative correlation with brain mitochondrial membrane potential and might play scathing role with mitochondrial function.Chapters 3:Protective test of liver mitochondrion in the early stage of sepsis.24 SD rats were randomly divided into three groups:saline control group,LPS group and insulin therapy group.In the early stage of sepsis,continue insulin infusion (0.25U/kg.h) could significantly reduce the over high blood glucose levels and maintain that in normal.We study the protective effects of continue insulin infusion on liver mitochondrion and its associated mechanism at 2h,6h,12h and 24h after intraperitoneal inject LPS.The results included that in insulin therapy group,the levels of serum ALT,AST,TNF-αand IL-6 levels all decreased significantly compared with LPS group.Besides,compared with control group,membrane potential of liver mitochondrion decreased significantly both in LPS group and insulin therapy group,while membrane potential of liver mitochondrion in insulin therapy group were significantly higher than that in LPS group.The activities of SOD in liver mitochondrion both in LPS group and insulin therapy group were significantly higher than that in control group.Compared with the LPS group,the activities of SOD decreased significantly in insulin therapy group.Minor damage of liver mitochondrion was found in LPS group,compared with that,the morphology of liver mitochondrion was improved in insulin therapy group.The inversely correlated relationship was found between membrane potential of liver mitochondrion and serum ALT,AST levels,serum TNF-α,IL-6 levels and blood glucose levels.Through the research,we can learn that in the early stage of septic rats,functional injuries of liver mitochondrion are obviously,but the damage of liver mitochondrial ultramicrostructure is minor.Liver mitochondrial injury in the early stage of septic rats is reversible.Continue insulin infusion can protect liver mitochondrion.This effect may work by attenuating inflammatory reaction,decreasing oxygen stress level and blood glucose level.