The Prognostic Value Of Ischemia-modified Albumin And The Salutary Effects Of PARP-1 Inhibition On Hepatic Mitochondrial Injuryin Sepsis

BackgroundSepsis is the presence or presumed presence of an infection accompanied by evidence of a systemic response called the systemic inflammatory response syndrome.Severe sepsisrefers to sepsis patients with organ dysfunction,poor tissue perfusion or hypotensive state.It is a major source of morbidity and mortality throughout the world.To improve clinical outcomes with sepsis,patients who are at risk of short-term mortality should be identified and the clinical decision-making process should be optimized.One of the most important events leading to morbidity and mortality in patients with severe sepsis is the development of global tissue hypoperfusion and following oxidative stress damage.Tissue hypoperfusion and hypoxia could lead to mitochondria dysfunction and generalized reactive oxygen species(ROS)production,then further extensive oxidative stress damage to cells and tissues.Sepsis-induced ischemia and oxidative stress damage conceivably play a key role in the pathogenesis of organ dysfunction and are believed to be the prelude to the development of multiple organ failure and death.Early recognition of the above progress may be helpful for timely clinical intervention.Ischemia and the generation of ROS can alter the ability of the N-terminal region of the albumin molecule to bind transitional metallic ions such as cobalt,copper and nickel.This modification of albumin,called ischemia-modified albumin(IMA),can be measured by albumin cobalt binding assay.IMA was first measured to detect early myocardial ischemia before the onset of myocardial necrosis;since then,increasing evidences showed elevated IMA levels in most patients with ischemic events(e.g.cerebrovascular occlusion,pulmonary ischemia,gastrointestinal ischemia,and muscle ischemia)and conditions that are potent producers of free radicals(e.g.liver cirrhosis,renal failure and advanced cancers).IMA is a marker of oxidative stress and ischemia-reperfusion in different clinical conditions and has prognostic value in patients with acute coronary diseases,end-stage renal diseases or their preoperative status.ObjectivesIn patients with sepsis,IMA level can be used to quantify the degree of ischemia and oxidative stress and may be a biomarker of outcome and death.However,there is a dearth of data regarding the prognostic value of IMA levels in such patients.Here,we prospectively collected data on patients with severe sepsis admitted to ICUs in a teaching hospital and try to determine the following hypothesis:1.The serum IMA level could predict short-term(28-day)mortality.2.The best cut-off value of serum IMA level to predict short-term mortality.3.The diagnostic efficiency comparison between serum IMA and adjusted IMA level for short-term mortality.Methods1.Setting and patientsA prospective observational cohort study was performed from April 2014 to October 2014 in the ICUs of Qilu Hospital of Shandong University.The study was approved by the Ethics Committee of Qilu Hospital of Shandong University.Severe sepsis is defined according to the international guidelines of the Surviving Sepsis Campaign.Subjects were excluded if they 1)had other ischemic diseases,such as acute coronary syndrome,acute ischemic cerebrovascular diseases,acute pulmonary embolisms,or acute lower-limb arterial or venous embolisms,aortic dissection;2)had preexisting malignant arrhythmia or cardiac arrest;3)had an undrained source of surgical sepsis;4)were<18 years,with AIDS or pregnant;5)had extremely low or high serum albumin levels(<20 or>55g/L);or 6)died within 24 hr or had a length of stay<72 hr,or had do-not-resuscitate orders.All patients received appropriate treatments according to the international guidelines of the Surviving Sepsis Campaign.Within 24 hr after the onset of severe sepsis,data on patientsdemographics,sources of infection,laboratory results,and co-morbidities were collected,and the Acute Physiology and Chronic Health Evaluation II(APACHE II)scores and Sepsis-related Organ Failure Assessment(SOFA)scores were calculated.Only one primary infection source for each patient was recorded.The primary outcome was 28-day mortality.2.MeasurementsSamples of 5 mL of peripheral venous blood were collected from patients within 24 hr of the onset of severe sepsis.The serum was separated by centrifugation at 1620 g for 15 min,then decanted and measured for IMA immediately by a commercially available albumin cobalt test kit according to the manufacturer's instructions and using their reagents and equipment.Albumin-adjusted IMA was calculated as(individual serum albumin content/median serum albumin content of the population)×IMA value.IMA level might be affected by the presence of very low albumin level(<20g/L),and this formula overcomes this interference(median serum albumin content for of each group of subjects was used separately).3.Statistical AnalysisSPSS 16.0 was used for data analysis.Data for categorical variables are expressed as a percentage and continuous variables as mean±SD.Chi-square or Fisher's exact test(two-tailed)was used to compare categorical variables.Unpaired Student's t test was used to compare continuous variables.The variables associated with mortality at P<0.05on univariate analysis were included in multivariate,backwards,stepwise,logistic regression analysis to identify independent risk factors associated with 28-day mortality.Results are presented as odds ratios(OR)with 95%confidence intervals(95%CI)and P-values.The area under the receiver operating characteristic(ROC)curve(AUC;with 95%CI)for IMA level as a prognostic factor,the sensitivity and specificity of IMA,and the optimal cut off value maximizing sensitivity and specificity were estimated by ROC curves.Correlations were examined by Pearson's correlation coefficient.Kaplan-Meier analysis and log-rank test were used to compare day-28 survival.A P<0.05 was considered statistically significant.Results1.Patient demographics and infection sourcesA total of 117 subjects with severe sepsis were enrolled(61.5%male;mean age 57.7±18.4 years).The 28-day mortality was 24.8%(29 non-survivors).The two main primary infection sources leading to severe sepsis were the lungs and abdomen/pelvis.2.Ability of IMA and albumin-adjusted IMA levels to predict 28-day mortalityWe generated ROC curves for IMA and albumin-adjusted IMA levels.Both IMA and albumin-adjusted IMA levels were strong predictors of 28-day mortality as indicated by an area under the ROC curve of 0.742(P<0.001)and 0.627(P = 0.040),respectively.The AUC for IMA was 0.742(95%Cl:0.638-0.847),and the optimal cutoff was determined to be 110 U/ml,with sensitivity 0.517 and specificity 0.886.3.Factors predicting mortalityOn univariate analysis,non-survivors more often had septic shock,acute kidney injury,invasive mechanical ventilation and renal replacement therapy than survivors(all P<0.01).IMA and glucose were higher for non-survivors than survivors,as was the Glasgow coma scale and APACHE ? and SOFA scores(all P<0.05).Albumin level was lower for survivors than non-survivors(P<0.05).On multivariate logistic regression,independent risk factors for death were IMA level(OR 2.104,95%CI 1.032-4.288,P = 0.041)and APACHE ? scores(OR 1.166,CI 1.078-1.261,P<0.001).IMA level and APACHE ? scores were not correlated(r=0.064,P= 0.495).4.Kaplan-Meier analysis of survival with different IMA levelsAccording to the optimal cutoff we determined and from previous empirical evidence,IMA was divided into 3 levels:low(<100U/ml),high(100?110U/ml)and very high(?110U/ml).On Kaplan-Meier analysis,28-day survival differed by the levels(P<0.001).Mortality at 28 days was higher with very high IMA level than both low and high IMA levels(all P<0.05).Conclusions1.The IMA level is increased in critically ill patients with severe sepsis.The IMA 9 level is a useful predictor of short-term mortality with severe sepsis.It had strong predictive power for 28-day mortality and was found an independent risk factor for early death.2 As an useful predictor of short-term mortality with severe sepsis,the optimal cutoff of IMA level was determined to be 110 U/ml.Physicians should be aware of the high death risk in this population with IMA level ?110U/ml,because early and timely clinical intervention is needed to improve the prognosis.3.Albumin-adjusted IMA levels did not perform better than IMA level for the diagnosis of early death,thus using albumin to adjusted IMA is not necessary.BackgroundSepsis refers to systemic inflammatory response syndrome due to infection,which can cause hypoperfusion and cell metabolism disorders,even lead to multiple organ failure or death.The liver is one of the organs that are easily injuried in sepsis.As the largest organ,liver is responsible for metabolism,detoxification,secretion of bile,immune defense,coagulation and other important functions.These important physiological functions make the liver as a critical organ for host survival following sepsis.Recent studies have shown that mitochondrial injury may play an important role in the pathophysiology of sepsis-induced organ failure.As the intracellular energy transfer organelles,mitochondria are the main sites for adenosine triphosphate(ATP)production and provide energy for a variety of cell life activities.The mitochondrial oxidative phosphorylation(OXPHOS)process is performed by two electron transport chains consisting of the complex ?-? on the mitochondrial inner membrane.Any damage in this process can lead to reduced ATP production and metabolic disorders of cells or organs.Targeted therapy for mitochondrial injury may be a valuable method of early targeted intervention for sepsis.The following molecular regulatory pathways may be the main mechanism of mitochondrial injury in patients with sepsis:(1)mitochondrial biogenesis pathway:Stable and effective mitochondrial biosynthesis is essential to maintain normal morphology and function of mitochondria.This process is regulated by peroxisome proliferator-activated receptor-y coactivator 1-?(PGC-1a),which is a key player that orchestrates mitochondrial biogenesis.It could activate transcription factors such as nuclear respiratory factors 1 and 2(NRF-1 and NRF-2),that upregulate nuclear production of mitochondrial proteins and subsequent expression of transcription factors such as TFAM(transcription factor A for the mitochondrion)that stimulate transcription of mitochondrial DNA;(2)oxidative stress regulation pathway:More than 90%of ROS in the cell is formed by electron transport chain electrons.In sepsis ROS in mitochondrion is formed largely,more than the clearance ability of the body.It can not only directly damage the membrane lipid,protein,DNA and other macromolecules in mitochondria,but also react with the electronic transport chain,causing reversible or irreversible inhibition of mitochondrial complex function,which further lead to mitochondrial injury.This process will lead to more oxidative stress products,and eventually form a vicious circle.The process is regulated by FOX03a,superoxide dismutase 2(SOD2),catalase(CAT)and other factors;(3)inflammatory regulation pathway:A large number of proinflammatory mediators and inflammatory factors are formed in sepsis,such as tumor necrosis factor ?(TNF-?),interleukin 6(IL-6)and so on,which produce mitochondrial toxicity,inhibit mitochondrial oxidative phosphorylation,reduce mitochondrial membrane potential and ATP production,reduce mitochondrial complex respiratory function and damage mitochondrial structure.In addition,high mobility group protein 1(HMGB1)is not only an important inflammatory regulatory factor,but also an important regulator of mitochondrial function and morphological maintenance.Interventions or drugs which can produce a beneficial effect on the above three pathways,are likely to play a target protection and improve mitochondrial function,reducing the occurrence and development of sepsis-induced organ injury and the mortality of patients.Poly(ADP-ribose)polymerase-1(PARP-1)isa protein-modified enzyme.Upon recognizing and binding to DNAstrand breaks,PARP-1 is activated and functions as the center ofstress responses,leading to DNA repair or cell death.Excessive activation of PARP-1 leads to intracellular depletion of nicotinamide adenine dinucleotide(NAD+)and ATP,thus resulting in cellular energy crisis,irreversible cytotoxicity and cell death.Pharmacologic inhibition of genetic deletion of PARP-1 has been shown to exert beneficial effects against inflammation cytokines,oxidant and free radical-mediated cell injury.It may play an important role in regulation and maintenance of tissue inflammation and oxidative stress.In addition,studies have shown that PARP-1 also had effect on the regulation of mitochondrial function.PARP-1 activation is prevalent in the development of sepsis.The beneficial effects of PARP-1 inhibition or knockout mentioned suggest that PARP-1 inhibitors may play a beneficial role in sepsis-induced mitochondrial injury.Although studies have shown that,PARP-1 inhibition or knockout had some potentially beneficial effects on sepsis-induced hemodynamic disorders and organ dysfunctions,whether sepsis-inducedhepatic mitochondrial injurybenefit from PARP-1 inhibition and the potential molecular regulatory mechanisms remain unknown.Objectives1.To investigate whether PARP-1 inhibition can alleviate sepsis induced hepatic mitochondrialinjury;2.To study the potential molecular mechanisms of the beneficial effect of PARP-1 inhibition on the hepatic mitochondrial injury,and to explore the role of mitochondrial biosynthesis,oxidative stress and inflammatory response regulations in these mechanisms.Methods1.Animal experiment:To induce endotoxemia and liver injury,maleC57BL/6 mice(sepsis,n=10)were treated with LPS(sigma,Lipopolysaccharides from Escherichia coli 055:B5,10mg/kg bw inPBS,i.p.),while the control animals(control,n=10)received the same volume of PBS.In order to ensure significant PARP-1 inhibition and following effects in mice,PARP-1 inhibition group(Sepsis+PJ34,n=10)mice were injected with PJ34(10mg/kg in normalsaline)intraperitoneally 3 hours before and 8 hours after modeling.Mice in sepsis group received intraperitoneal injection of the same amount of saline at the same time.24 hours after modeling,anesthetize animals to take specimens;2.After anesthesia,do the left ventricular puncture of the heart to take blood,detecting related indicators.Detect blood gas analysis and lactate levels with blood gas analyzer.Glutamic oxalacetic transaminase(AST)and glutamic-pyruvic transaminase(ALT)were measured using commercial kits;3.Real-timedetermination of mitochondrial respiratory capacity:After extraction of liver mitochondria from fresh tissue,detectreal-time respiratory capacity of mitochondrial electron transport chain complexes ?,?,? using the energy metabolism analyzer;4.Real-time mitochondrial membrane potential and ATP level:After extraction of liver mitochondria from fresh tissue,real-time mitochondrial membrane potential and ATP level were measured using commercial kits;5.The NAD+ level and PARP-1 activity were detected by commercial kits and spectrophotometry;6.Pathological test:Hematoxylin and eosin(HE)staining was used to observe the pathological changes of liver;7.Ultrastructural observation:Transmission Electron Microscope(TEM)was used to observe the ultrastructure of mitochondria;8.The serumlevels of TNF-?,IL-6 and HMGBlwere measured by commercial ELISA kits;9.Immunohistochemical staining:The expression of PGC1-? and 3-nitrotyrosine(3-NT)were observed by immunohistochemical staining;10.Realtime quantitative RT-PCR:Total RNA of the mice liver tissue was extracted by Trizol.The mRNA levels of TNF-??IL-6?HMGB1?FOXO-3a?Catalase?PGC1-??SIRT1?TFAM and NRF-1 were measured byrealtime PCR;11.Western blot:The expression of PGC1-??NRF-1?HMGB1?SOD2?4-hydroxynonenal(4-HNE)?3-NTwere measured by Western blot.Results1.LPS-induced sepsis model1.1.Mice in sepsisgroupshowed severe metabolic acidosis,hyperlipidemia,electrolyte disturbanceand liver damageCompared with control group,mice in sepsis group showed obvious hyperlactacidemia,acidosis,low HCO3-level,hyperkalemia and hypocalcemia,theserum level of AST and ALT were both significantly elevated(P<0.05).But the serum sodium level and hematocrit did not change significantly(all P>0.05).All mice in control group were alive(0%mortality).3 died in sepsis group(30%mortality).1.2.Mice in sepsisgroup showed significant decrease inmitochondrial respiratory function,ATP levels and mitochondrial membrane potential levelsCompared with control group,the respiratory capacity of mitochondrial complex I,complex ? and complex ? in sepsis group significantly decreased,the level of ATP and mitochondrial membrane potentialsignificantly decreased(P<0.05).1.3.Mice in sepsisgroup showed significant hepatic pathological damage and mitochondrial ultrastructural abnormalitiesThe liver of mice in sepsis groupshowed lobular structure disruption,hepatocyte swelling,obvious balloon-like changes,and visible spotty necrosis and inflammatory cell infiltration.TEM found significant mitochondrial swelling,unclearmitochondrial ridge and other mitochondrial internal structures,and the emergence of more mitochondrial autophagy.1.4.The expression of upstream factors in liver mitochondrial biosynthesis significantly decreased in sepsis groupCompared with control group,the PARP-1 activity in the liver of sepsis group mice significantly increased,the level of NAD+ significantly decreased(P<0.05).The mRNA level ofSIRT1,PGC1-? and NRF-1 significantly decreased,and the expression levels of PGC1-? and NRF-1 significantly decreased(P<0.05).Immunohistochemical staining showed that the expression levels of PGC1-?significantly decreased(P<0.05)and mainly expressed in the nucleus.1.5.The liver of sepstic mice showed significant oxidative stress injuryCompared with control group,the mRNA level of catalase in the liver of sepsis group significantly decreased,the mRNA level of FOXO-3a significantly increased(P<0.05).The protein expression level of the antioxidant enzyme SOD2 significantly decreased,while the protein expression level of 4-HNE and 3-NT significantly increased(P<0.05).Immunohistochemical staining showed that the expression levels of 3-NT significantly increased(P<0.05)and mainly expressed in the cytoplasm.1.6.Mice in sepsisgroup showed significant inflammation in the liverCompared with control group,theserum levels of TNF-a,IL-6 and HMGB1 in sepsis group significantly increased(all P<0.05),themRNA levels of TNF-?,IL-6 and HMGB1 significantly increased,the protein expression levels ofHMGB1 also significantly increased(P<0.05).2.The beneficial effects of PARP-1 inhibitor PJ-34 on sepsis mice2.1.PARP-1 inhibition significantly improved sepsis-induced lactic acidosis,hyperkalemia,low HCO3-level and decreased the levels of AST and ALTCompared with sepsis group,PARP-1 inhibition improved the lactic acidosis,the HCO3-level and the hyperkalemia,the levels of AST and ALT were significantly decreased(P<0.05).The mortality of PARP-1 inhibition group(10%)seemed to decrease compared with sepsis group(30%mortality),but without statistical significance(P>0.05).2.2.PARP-1 inhibition significantly improved sepsis-induced liver mitochondrial injuryCompared with sepsis group,PARP-1 inhibition significantly inicreased the respiratory capacity of mitochondrial complex ?,complex ? and complex ?,and significantly improved mitochondrial ATP level and mitochondrial membrane potential(P<0.05).2.3.PARP-1 inhibition can improve sepsis-induced liver mitochondrial structural damageCompared with sepsis group,there is no significant improvement of the pathological damage in PARP-1 inhibition group.However,TEM showed that the mitochondrial swelling and mitochondrial autophagy were alleviated,and themitochondrial internal structure became moreclear.2.4.PARP-1 inhibition significantly up-regulated the expression of upstream factors in liver mitochondrial biosynthesisCompared with sepsis group,the PARP-1 activity in PARP-1 inhibition group was significantly decreased,the level of NAD+ significantly increased(all P<0.05).The mRNA levels of SIRT1,PGCl-?,NRF-1 and TFAM significantly increased(P<0.05).The expression levels of PGC1-? and NRF-1 increased(P<0.05).Immunohistochemical staining also showed that the expression levels of PGC1-?significantly increased(P<0.05)2.5.PARP-1 inhibition significantly alleviated sepsis-induced oxidative stress in the liverCompared with sepsis group,the mRNA expression level of catalase and the expression level of SOD2 protein in PARP-1 inhibition group significantly increased,while the mRNA expression level of FOXO-3aand the expression level of 3-NT proteins significantly decreased(P<0.05).Immunohistochemical staining also showed that the expression levels of 3-NT proteins significantly decreased(P<0.05).The expression level of 4-HNE decreased,but there was no significant difference(P<0.05).2.6.PARP-1 inhibition significantly alleviated theinflammation in the liverCompared with sepsis group,the serum levels of TNF-a,IL-6 and HMGB1 in PARP-1 inhibition group were significantly lower(P<0.05).The mRNA levels ofTNF-?,IL-6 and HMGB1 significantly decreased,the protein expression levels of HMGB1 significantly decreased(P<0.05).Conclusions1.Mice with LPS-induced sepsis showed significant hepatic mitochondrial dysfunction and structural damage.The expression levels of the upstream factors of mitochondrialbiosynthesiswere significantly decreased,and significant oxidative stress injury and inflammatory response existed.2.PARP-1 inhibition could alleviate sepsis induced hepatic mitochondrial dysfunction and structural damage.It improvedthe respiratory capacity of mitochondrial respiratory complex,increased ATP production and membrane potential levels and reduced mitochondrial swelling and autophagy.3.PARP-1 inhibition mightplay a targeted effect of mitochondria through the positive regulation of mitochondrial biosynthesis,oxidative stress and inflammatory response,thereby improving thehepatic mitochondrial injuryin sepsis.