The Study On The Mechanism Of Notch Signaling Pathway Involved In Septic Encephalopathy

Diffuse brain dysfunction is a frequent complication of sepsis, usually defined as sepsis-associated encephalopathy (SAE). The incidence of encephalopathy secondary to sepsis is8%to70%. SAE is one of the most common encephalopathy in ICU. and is a severe complication of sepsis with high mortality rate. Although the mechanism of SAE is still largely unknown, accumulative evidences have indicated the involving of neuronal apoptosis. We found that SAE can lead to long-term memory impairment in the sepsis rats, especially on the hippocampus closely related with the spatial memory.This research is based on the Notch signal pathway as the main object, and explores the pathogenesis of SAE. Notch signaling is a highly conserved pathway, which plays a key role in regulating neuronal apoptosis. New research suggests that γ-secretase complexus and Notchl involved in the pathogenesis of several neurodegenerative diseases, such as Blzheimer’s disease and ischemic stroke. Moreover, Notch signal pathway inhibitor DAPT can significantly reduce brain ischemic injury caused by neuronal apoptosis and inflammation, related to Notch signaling pathway playing a regulatory role on the PARP-1activated. Recent studies found that PARP-1activated by their own division and nuclear apoptosis related factors, activation of the caspase pathway, lead to neuronal apoptosis.The CLP model was constructed in the experiment. After24h of CLP, the N1CD expression was significantly increased in CLP model, as well as hippocampal tissue inflammation in the rats, neuronal apoptosis, and cell death. DAPT suppressed hippocampal neuronal apoptosis and cell death induced by CLP in septic rats, as well as the expression of TNF-a,IL-1b,IL-6,PARP-1and Caspase-1. Moreover, DAPT significantly prevented NF-kB from nuclear transfer in CLP rats.In a word, Notch signaling pathway has been proved involved in brain dysfunction caused by sepsis, related to NF-kB nuclear transfer. The results can provide the strategy and new drugs’ target for the treatment of SAE. Part One The role of Notch signaling pathways in sepsis associated encephalopathyObjective:Sepsis associated encephalopathy (SAE) is the most common encephalopathy in the 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. Several studies have indicated γ-secretase complex and Notch1are involved in neurodegenerative diseases, and γ-secretase inhibitor, DAPT can prevent the brain from ischemia injury mediated by apoptosis and inflammatory. In this part, we selected the standard septic model of cecum ligation puncture (CLP). After septic encephalopathy model was set up successfully, we are trying to study the function of Notch signaling pathway in SAE and the DAPT neuro-protection of SAE.Methods:The rats were anaesthetized with pentobarbital (30mg/kg, intra-peritoneally). Under sterile surgical conditions, a2cm 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 twice 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. 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.59rats were categorized into four groups:(a) a sham group with normal saline (10mL/kg);(b) a sham group with DAPT (10μmol/kg);(c) cecal ligation and puncture (CLP) with normal saline (10mL/kg); and (d) CLP with DAPT (10μmol/kg), and every group was divided into two subgroups. Rats in first subgroup for western blot assay were sacrificed and their brains were harvested24h later. The hippocampus was quickly isolated and stored at-80℃. The rats in the second subgroup were given normal saline and ceftriaxone every day and permitted normal eating and drinking. We would evaluate new object recognition test (7days after operation).Results: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 reduced obviously (3days,56.3%;4-7days,37.5%). Neurologic reflex scores decreased in24h in the CLP group and recover gradually from4to7days after operation. The new object recognition test suggested that the CLP group impaired learning and memory function. Sepsis can increase the expression of hippocampal Notch receptor intracellular domain (NICD), and DAPT can significantly decrease the level of NICD and PARP-land rescue cognitive impairment caused by SAE.Conclusion:CLP models simulate the typical septic clinical manifestations, sepsis associated encephalopathy (SAE) model is established successfully by evaluating neurologic reflex scores, behavior trials. The present results firstly demonstrated the significant increased expression of NICD in the hippocampus24hours after CLP, indicated that the Notch signaling pathway may take part in the progress of SAE and the neuroprotective effect of DAPT on memory impairment in septic rats. Part Two:The mechanism of Notch signaling pathway involved in septic encephalopathyObjective:In this experimental study, we build experimental Sepsis using a Cecal Ligation and Perforation Mouse Model, focuses on the possible pathogenesises by which the Notch signaling pathway is involved in the sepsis associated encephalopathy, and the interventional effect of y-secretase inhibitors by blocking Notch1signaling pathway in septic encephalopathy rats.Method:59SD rats were randomly divided into four groups, namely sham group, sham+DAPT group, CLP group and CLP+DAPT group.24hours after CLP or sham-operation, all rats were sacrificed. The expression of TNF-a, IL-1b, IL-6, PARP-1and Caspase-1were detected with Westen blot and ELISA analysis in the removed hippocampal tissue. Neuronal apoptosis and neuronal death were observed by TUNEL method and HE, respectively.Result:Our studies show that in experimental animals, the level of TNF-a, IL-1b. IL-6. PARP-1and Caspase-1expression increased significantly in CLP group compared with sham and sham+DAPT group, despite the increase was inhibited in sham+DAPT group. Neuronal apoptosis and neuronal death were observed by TUNEL method and HE, respectively. In the sham group and sham+DAPT group hardly find HE positive cells. The neuronal cell death increased significantly in the CA1of the rat hippocampus after the CLP surgery. However, compared with the CLP group, HE staining positive cells in the CLP+DAPT group decreased significantly. In addition, the neurona! apoptosis was detected by TUNEL. In the CLP group. TUNEL-positive cells increased significantly, but in the sham group barely detected TUNEL-positive cells. Number of neuronal apoptosis in CLP+DAPT group was significantly greater than sham+DAPT group. Compared with CLP group, the number of neuronal apoptosis in the first two groups reduced significantly. In septic rats, the hippocampus tissue inflammation. neuronal apoptosis and neuronal death increased significantly. DAPT can significantly reduce the hippocampus neuronal apoptosis and death, the release of TNF-a, IL-1b, IL-6.Conclusion:In this study, we confirms that the Notch signaling pathway by activating the PARP-1degradation, leading to caspase-dependent neuronal apoptosis, y-secretase inhibitor DAPT inhibition of hippocampal neurons PARP-1degradation, inhibition of caspase-dependent neuronal apoptosis. DAPT by inhibiting the CLP sepsis in the rat hippocampus neuron apoptosis and inflammatory reaction improve sepsis encephalopathy Part Three:The regulation of Notch on septic encephalopathy through NF-kB signaling pathwayObjective:The key transcription factor NF-kB which is located at the nucleus is involved in inflammation, also widely distributed within the cells of nervous system, regulating the inflammation and apoptosis in neuronal cells. Several previous studies indicated that NF-kB signaling pathway also play an important role in septic inflammation of the nervous system. Here, using the cecal ligation and puncture (CLP) model, we investigated whether the NF-kB signaling pathway is involved in Notch signaling mediated septic encephalopathy.Methods:Adult Sprague-Dawley rats were randomly divided into four groups (n=6), namely Sham group, Sham+DAPT group, CLP group, CLP+DAPT group. The subjects were peritoneally injected DAPT (10mmol/kg) or saline (10mL/kg),30min before operation.Saline (3mL/100g) and Ceftriaxone (30mg/kg) were subcutaneously injected per6h from postoperative day. Rats were sacrificed after24h.Hippocampal slices were obtained from SD rats that were stored in liquid nitrogen. The expression of NF-kB protein was evaluated in groups by Western blot and EMUSA analysis.Results:in sham group rats, nuclear factor NF-kB was expressed in the cytoplasm, and transferred from cytoplasma to nucleus in the cecal ligation and puncture (CLP) model.Expression of NF-kB, especially P65. increased significantly in nucleus in CLP group compared with Sham. Expression of NF-kB, increased significantly in nucleus in CLP group compared with that of sham plus DAPT group, but does not include P65indicated that the level of p65,p50and RelB expression decreaded after the intervention of DAPT. Comparison with sham group, phosphorylated IKK-β expression was significantly increased in CLP group. However, the intervention of DAPT inhibited phosphorylated IKK-β expression in the cecal ligation and puncture (CLP) model. Similarly, the intervention of DAPT also inhibited p65phosphorylation. which may be associated with the decreased activity of IKK-β Correspondingly P65DNA-binding activity would decrease inevitably. Conclusions:In this study, we showed nuclear factor NF-kB transferred from cytoplasma to nucleus in the cecal ligation and puncture (CLP) model. The intervention of DAPT (Notch-specific inhibitor) inhibited Notch target gene expression, NF-kB transferred from cytoplasma to nucleus and DNA-binding activity. Combined with analysis of previous reports, hippocampal neurons inflammation and apoptosis in sepsis induced by the transcriptional activation which was NF-kB dependent, probably is one of the reasons that lead septic encephalopathy. Inhibition of Notch signaling pathway may be an efficient way to treat septic encephalopathy.