Protective Effects Of Continuous Norepinephrine Infusion In Early Septic Rats

ObjectiveSepsis is infection plus systemic inflammatory response syndrome,its incidence andmortality rates are high. Septic shock is the major complications in patients withsepsis,often leading to multiple organ failure,and even death. In septicshock,catecholamines(epinephrine、norepinephrine and dopamine)drugs is important forincreasing blood pressure and stabling mean arterial pressure. In2008,the internationalsepsis guide suggest norepinephrine is futher treatment measures after fluid resuscitationand dopamine treatment failure. Recent studies have found that norepinephrine can beused not noly to increasing mean arterial pressure,but also can reduce the inflammatorycytokines and oxidative stress levels,improve microcirculation and mitochondrialrespiratory function,but is still controversial. With the understanding of sepsis,currentlyconsidered sepsis leads to mitochondrial dysfunction mechanisms includemicrocirculatory disorders、 inflammatory damage、 oxidative stress、 nitric oxideoverexpression and mitochondrial DNA damage. Thus,norepinephrine on criticalillness(especially sepsis) patients have certain protective and restorative effect. In addition,the liver is the vital organs of substance and energy metabolism, and in the early stage ofsepsis is vulnerable. The animal experimental study continuous infusion of low dosenorepinephrine intervention on early sepsis rats,including serum inflammatory cytokines,liver oxidative stress and mitochondrial respiratory function and its related mechanisms,and to identify the appropriate dose for norepinephrine, it can supply new method andthought for reduce sepsis incidence of serious complications and mortality rate.Methods1. Animal preparationThe septic model was set up by receiving lipopolysaccharide10mg/kg byintraperitoneal injection. Fourty four SD rats were randomly divided into five groups (every rat was performed with an external jugular vein catheterization a day beforeintraperitoneal injection): saline control group (n=8), LPS group (n=8) and norepinephrinetherapy group (low dose、dose、high dose,n=3*8). Saline control group animals receivedintraperitoneal injected with0.9%saline10ml/kg, and then received an infusion of0.9%saline1ml/h. LPS group animals were intraperitoneal injected with lipopolysaccharide10mg/kg, and then received an infusion of0.9%saline1ml/h.therapy group animalsreceived an infusion of norepinephrine solution1ml/h after being intraperitoneal injectedLPS10mg/kg. The dose of norepinephrine are0.06、0.3、0.6ug/kg/min.2. Measurements and methods(1) General state of animalsRectal temperature, heart rate and breathing rate were observed before intraperitonealinjection and at2nd hour,6th hour,12th hour,24th hour after intraperitoneal injection.(2) Markers of inflammatory reactionAt2nd hour and6th hour after intraperitoneal injection, serum TNF-αand IL-6wereassessed by ELISA.(3) Markers of biochemistryLiver function: Serum ALT and AST were detected by automatic biochemistry analyzer at24th hour after intraperitoneal injection.(4) Markers of liver mitochondrial injury①At24th hour after intraperitoneal injection, mitochondrion of liver were isolated bydifferential centrifugation.Swelling and membrane potential of liver mitochondrion in ratswere analyzed by FCM.②At24th hour after intraperitoneal injection,Rat liver isocitrate dehydrogenase activityand mitochondrial pyruvate dehydrogenase activity detect by ELISA.③At24th hour after intraperitoneal injection, mitochondrial oxidative stress level(SOD、MDA、NO and iNOS) of liver in rats were detected by chromatometry.④At24th hour after intraperitoneal injection, liver mitochondrion were observedthrough electronic microscopy and the degree of injury in liver mitochondrion wereanalyzed by Flameng semiquantitative evalution of ultrastructure.3. Statistical analysisThe datas were analyzed by SPSS13.0. All results were reported as mean±standarddeviation(±s). Differences between groups were determined by ANOVA. If there was statistical significance and homogeneity of variance between groups, Least-significantdifference (LSD) was used. If there was statistical significance and heterogeneity ofvariance, we used Games-Howell. P values<0.05was considered significant.Results1. General state of animalsAll animals in endotoxin group and norepinephrine therapy group showed septicsymptoms and signs.2. Markers of inflammatory reaction(1) Serum TNF-αa、Serum TNF-α level after2h of intraperitoneal injection of LPS(or saline)：Compared with the normal control group,endotoxin group and intervention group (lowdose and middle dose group) serum levels TNF-α increased significantly(P=0.000,P=0.001,P=0.042);compared with the endotoxin group,the interventiongroup(middle dose and high dose group)serum TNF-α levels were significantlyreduced(P=0.037,P=0.039).b、Intraperitoneal injection of LPS (or saline) after6h, serum TNF-α level not detected.(2) Serum IL-1βa、Serum IL-1β level after2h of intraperitoneal injection of LPS(or saline)：Compared with the normal control group,endotoxin group and intervention group(low dose group) serum levels IL-1β increased significantly (P=0.07,P=0.04);comparedwith the endotoxin group,the intervention group(middle dose group)serum IL-1β levelswere significantly reduced(P=0.02).b、Serum IL-1β level after6h of intraperitoneal injection of LPS(or saline)：Compared with the normal control group,endotoxin group serum levels IL-1βincreased significantly (P=0.033);compared with the endotoxin group,the interventiongroup(middle dose group)serum IL-1β levels were significantly reduced(P=0.041).(3) Serum IL-10a、Serum IL-10level after2h of intraperitoneal injection of LPS(or saline)：Compared with the normal control group,endotoxin group and intervention group(low dose、middledose and high dose group) serum levels IL-10increased significantly(P=0.001， P=0.000， P=0.000);compared with the endotoxin group,the interventiongroup(middle dose group)serum IL-10levels were significantly reduced(P=0.032). b、Serum IL-10level after6h of intraperitoneal injection of LPS(or saline)：Compared with the normal control group,endotoxin group and intervention group(low dose、middledose and high dose group)serum levels IL-10increased significantly(P=0.001，P=0.000，P=0.000);compared with the endotoxin group,the interventiongroup(middle dose group)serum IL-10levels were significantly reduced(P=0.048).3. Markers of biochemistrySerum ALT and AST in endotoxin group were significantly higher than that in controlgroup (P=0.048，P=0.028). Compared with endotoxin group, serum ALT and AST havedecreased trend,but there no significant difference.4. Markers of liver mitochondrial injury(1) Swelling and membrane potential of liver mitochondrionCompared with control group, membrane potential of liver mitochondrion decreasedsignificantly both endotoxin group and intervention group (low dose)(P=0.013，P=0.008).Membrane potential of liver mitochondrion in intervention group(high dose group) weresignificantly higher than that in endotoxin group (P=0.03).The difference wasn’t statistically significant in Swelling of liver mitochondrionamong five groups (P>0.05).(2) Liver pyruvate dehydrogense (PDH) activity and liver mitochondrial isocitratedehydrogense (IDH) activityCompared with control group, liver PDH activity decreased significantly bothendotoxin group and intervention group (low dose)(P=0.009，P=0.009); compared withendotoxin group,liver PDH activity in intervention group(high dose group) weresignificantly higher (P=0.003).Compared with control group, liver mitochondrial IDH activity decreasedsignificantly in endotoxin group(low dose)(P=0.015); compared with endotoxingroup,liver mitochondrial IDH activity in intervention group(high dose group) weresignificantly higher (P=0.027).(3) Oxidative stress level in liver mitochondrionCompared with the control group, the activities of liver mitochondrion iNOS、MDAand NO in each group have increased trend,and compared with the intervention group,theintervention group have decreased trend,but all difference were not statistically significant. The activities of SOD in all group was not statistically significant.(4) Morphology of liver mitochondrionThe changes of liver mitochondrial ultrastructure was not obviouse and nosignificant difference in its semiquantitative evaluation scores among three groups(P>0.05).Conclusions1. There are obviouse injuries of liver function、inflammatory injury and impaired mitochondrialfunction in the early stage of septic rats. But the injury of liver morphology is slight.2. Continuous norepinephrine infusion have a certain degree of protection and restorationfunction in early stage of septic rats. This effect may work by attenuating inflammatoryreaction、enhancing tricarboxylic acid cycle key enzyme activity and mitochondrialmembrane potential level. And this effect may have no relationship with oxidative stressin the early stage of septic rats.