The Role Of Omi/Htra2Signaling Pathway In Sepsis Associated Encephalopathy

The morbidity and mortality of sepsis is very high. Sepsis-associated encephalopathy(SAE) is diffuse brain dysfunction caused by sepsis. The nervous system is one of the earliest organs involved in sepsis. SAE incidence rate is8%-70%, and it is the most common encephalopathy in intensive care unit (ICU). The mortality of SAE is very high, but the pathophysiological mechanism is not clear, the current studies have found that brain microvascular endothelial cell dysfunction, blood-brain barrier (BBB) damage, inflammatory cell infiltration, inflammatory mediators, decreased cerebral perfusion, brain microvascular dysregulation, astrocytes and neurons dysfunction, neurotransmitters disorders, mitochondrial dysfunction, apoptosis, oxidative stress and calcium disorders are involved in SAE pathogenesis. And there are no effective therapies for SAE.Studies have confirmed that mitochondrial dysfunction is involved in SAE. Mitochondria membrane potential is destructed, Mitochondrial respiratory chain is influenced, Mitochondrial disfunction can cause cell energy supply shortage and induce cell apoptosis. The studies have demonstrated that Omi/HtrA2is a kind of oligomeric serine protease. It has been confirmed that Omi/HtrA2can regulate apoptosis in a variety of pathological conditions (cardiac ischemia/reperfusion injury, the renal dysfunction caused by cisplatin, cerebral ischemia/reperfusion injury, etc.), and several studies have confirmed that the Omi/HtrA2is involved in a variety of nervous system diseases (such as huntington’s disease, cerebral ischemia/reperfusion and status epilepticus, etc.). In this study we would establish SAE rat models. We would be trying to study hippocampus cell apoptosis during sepsis, and Omi/HtrA2signaling pathways (Omi/HtrA2concentration in the mitochondria and cytoplasm, and XIAP concentration, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentrations in the cytoplasm), and Omi/HtrA2protease inhibitor(ucf-101) neuroprotective effect. We would be trying to demonstrated Omi/HtrA2signaling pathway is invovled in SAE, It would help to discover a novel therapeutic target and new drug for SAE. Part One Establishment of a rat model of sepsis associated encephalopathyObjective:Sepsis associated encephalopathy (SAE) is the most common encephalopathy in intensive care unit (ICU). The mortality of SAE is very high, but the pathophysiological mechanism is not clear and no effective treatment has been confirmed. Ideal animal models are needed in basic research urgently. In this part, we selected the standard septic model of cecum ligation puncture (CLP) and septic encephalopathy model was considered to be set up successfully by evaluating the behavior trials, neurologic reflex scores and hippocampal pathology which can be used in the next experiments.Methods:Animals were anesthetized with4%Chloral hydrate (1ml/100g, intraperitoneally). Under sterile surgical conditions, a2-cm abdominal incision was made along the ventral surface of the abdomen to expose the cecum, which was then ligated below the ileocecal junction with no bowel obstruction. The cecum was punctured once with an18-gauge needle, and the fecal contents were allowed to leak into the peritoneum by gently squeezing the cecum. The bowel was then returned to the abdomen and the abdominal cavity was closed. The sham-operated rats were submitted to laparotomy, and the cecum was manipulated but neither ligated nor punctured. All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g body weight) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation. In the first experiment, blood pressure, heart rate and anus temperature were measured at baseline,6h,12h and24h after operation. At the same time, we measured blood lactic acid level. We also observed hippocampal pathology24h post-operation (n=6). In the second experiment, rats (n=16) were given normal saline and ceftriaxone every day and allowed normal eating and drinking. We would evaluate neurologic reflex scores, survival rate, new object recognition test (7day after operation) and Morris water maze test (4,7days after operation).Results:The septic rats appeared crouching, pilomotor, hemiplegia or seizures. Significant decreased mean arterial pressure, increased heart rate, deteriorated neurological reflexes together with increased blood lactate levels suggested the successful induction of sepsis in the present study. Survival rate of the CLP group decreased obviously (3days,43.75%;4-7days,37.5%). Hippocampal H&E staining showed most neurons were shrunken and stained dark in the CLP group, significantly in dentate gyrus (DG) area. Behavior trials (Morris water maze test and new object recognition test) suggests that the CLP impaired learning and memory function.Conclusion:CLP models simulate the typical septic clinical manifestations, sepsis associated encephalopathy (SAE) model is established successfully by evaluating neurologic reflex scores, behavior trials and hippocampal pathology, which can be used in SAE mechanism study. Our study demonstrated hippocampus damage may be involved in SAE. Part Two Neuro-protective effect of Omi/HtrA2protease inhibitor (Ucf-101) on sepsis associated encephalopathyObjective:There are no effective treatments for SAE. It has been demonstrated that Omi/HtrA2protease inhibitors (Ucf-101) can improve a variety of pathological conditions (such as huntington’s disease, cerebral ischemia/reperfusion and status epilepticus, etc.). We are trying to study Ucf-101neuro-protection of SAE.Methods:The animals were categorized into four groups:(1) sham group plus normal saline (10mL/kg);(2) sham group plus Ucf-101(10umol/kg);(3) cecal ligation and puncture (CLP) plus normal saline (10mL/kg); and (4) CLP plus Ucf-101(10umol/kg). All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation. In the first experiment,24hours later, animals were sacrificed and their brains were harvested. Samples were used for analysis of inflammatory cytokine and oxidative stress (n=6), evaluation of apoptosis and pathologic changes (n=6). In the second experiment, All rats(n=16) received a intraperitoneal injection of saline solution (lOmL/kg) or Ucf-101(10umol/kg) every day, and was allowed eating and drinking normally, we would evaluate weight, neurologic reflex scores, survival rate, new object recognition test (7days after operation). Morris water maze test were also evaluated (4,7days after operation).Results:CLP resulted in a poor survival rate, hippocampal oxidative injury, cell apoptosis and cognitive dysfunction as well as elevated TNF-a level and IL-6level, increased weight loss. Ucf-101could significantly inhibit cell apoptosis, reduce TNF-a and IL-6levels, MDA and MPO levels, elevated GSH level, slightly reverse CAT activities in the brain and attenuate this CLP effect on cognitive dysfunction. In addition, the survival rate and survival time was significantly improved by treatment with Ucf-101.Conclusion:The present results demonstrated that sepsis can cause hippocampal inflammation, oxidative stress injury, apoptosis, and cognitive memory dysfunction. Ucf-101can significantly reduce the hippocampal cell apoptosis, inflammation and oxidative stress damage and improve cognitive function. So Ucf-101might have neuro-protective effect and Omi/HtrA2could be a new target for SAE. Part Three Omi/HtrA2signaling pathways involved in sepsis associated encephalopathyObjective:Omi/HtrA2is involved in a variety of nervous system disease.This study aims to study whether Omi/HtrA2signaling pathways were involved in SAE.Methods:The animals were categorized into four groups (n=6):(1) sham group plus normal saline (10mL/kg);(2) sham group plus Ucf-101(10umol/kg);(3) cecal ligation and puncture (CLP) plus normal saline (10mL/kg); and (4) CLP plus Ucf-101(10umol/kg). All rats underwent surgical manipulation, received a subcutaneous injection of saline solution (3mL/100g) plus antibiotics (ceftriaxone at30mg/kg) every6h for resuscitation.24hours later, animals were sacrificed and their brains were harvested. Samples were used for analysis of Omi/HtrA2concentration in the mitochondria and cytoplasm, and XIAP concentration, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP and cytochrome c concentrations in the cytoplasm.Results:Omi/HtrA2is released into cytoplasm from mitochondria in the CLP group. XIAP expression decreased significantly, cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentration increased significantly. Ucf-101inhibited the translocation of Omi/HtrA2, reduced degradation of XIAP, and reduced cleaved Caspase-3, cleaved Caspase-9and cleaved PARP concentrations. This study also found that cytochrome c is released from mitochondria to the cytoplasm in CLP group and Ucf-101was unable to prevent the effect.Conclusion:This study demonstrated that in sepsis Omi/HtrA2is released from mitochondria to cytoplasm, and it could degrade XIAP and activate the downstream of the apoptosis pathways. Omi/HtrA2mediated signal pathway is involved in sepsis associated encephalopathy, so it could be a new target for SAE.