| Inflammation process in brain is an important event accompanied with a series of neurodegenerative disorders. The role inflammation played in these disorders has been controversial. Inflammation was usually taken as benefit against extrinsic stimulus, on the contrary, excessive inflammation can also be destructive. Increasing more evidences indicated that inflamation can be a driving force in promoting these neurodegenerative disorders. Pheripheral cytokines or inflammatory factors were unlikely to impact on brain parenchyma due to the existence of blood-brain barrier (BBB). Howerver, prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) derived from arachidonic acid were indicated to get through the BBB and permeated into hypothalamus regions and finally induced pyresis or anorexia. Brains cortex and hippocampus are important regions related with cognition and memory, they are closely related to several neurodegenerative disorders such as Alzheimer's disease (AD). Therefore, it is possible that PGE2 and/or LTC4 may also act on cortex and hippocampus and finally lead to downstream pathological changes in these regions.It was remained elusive that the PGE2 and LTC4 production in cortex and hippocampus respectively in a systemic inflammatory model. Therefore, in the first chapter, lipopolysaccharide (LPS) induced inflammation in rats and cycloxygenases (COXs) inhibitor indomethacin (INDO) were employed. The regulation role of PGE2 on early AD-related pathology characterized by 3-nitrotyrosine (3-NT) formation.Tumor necrosis factor-α(TNF-α) is an important cytokine produced within brain tissues following inflammatory challenge. TNF-αis involved in acute and chronic inflammation found in neurodegenerative disorders including AD. It was found that TNF-αsignaling impacted onβ-site amyloid precursor protein cleaving enzyme 1 (BACE1) regulation, suggesting TNF-αsignaling may promote AD development. In the second chapter, TNF-αantagonist etanercept (ETC) was employed to investigate the regulation role of TNF-αon BACE1 expression.In the present research, several biological systems both in vivo and in vitro were employed to address the potential impact of PGE2 and TNF-αon related pathological events in brain. It was expected to find the mechanisms underlying inflammation impact on brain pathological changes, and further to discover therapeutic targets for neurodegenerative disorders1. PGE2 and LTC4 synthases expression changes and 3-nitrotyrosine formation in LPS-challenged rats brain cortex and hippocampusTo address the potential influence of PGE2 on related pathological changes in brain cortex and hippocampus, rats were challenged by LPS (2 mg/kg, i.p.) injection. Within 24 h injection of LPS, mPGES-1 expression in cortex and hippocampus was up-regulated in a time-dependent manner. LPS-induced fever was inhibited by cycloxygenases inhibitor indomethacin (2 mg/kg, i.p. INDO) injection, suggesting LPS induced PGE2 production in these brain regions, the LPS-challenged inflammatory model has been established. On the other side, LPS challenge induced a transient up-regulation of LTC4S but had no effect on mGST3 expression. Nitric oxide synthases (NOSs) and Nicotinamide-adenine dinucleotide phosphate oxidases (NOXs) are major enzymes catalyze NO and ROS production in vitro, respectively.1 d after LPS challenge, eNOS, iNOS and NOX4 were up-regulated in rats hippocampus, as well as NO and ROS production in tissues. NO and ROS lead to the peroxynitrite (ONOO") production, which further induced 3-nitrotyrosine (3-NT) formation.3-NT formation was suggested to be closed related with AD pathology. Immunohistochemistry analysis revealed that 3-NT level in hippocampus was up-regulated 7 d after LPS challenge, indicating inflammation may progress AD process in an early stage.3-NT is suggested to be involved in cell apoptosis. The apoptosis related proteins caspase-8 and Bax in hippocampus were determined. As results shown, LPS up-regulated caspase-8 and Bax expression 7 d after injection, suggesting 3-NT formation may ultimately induced apoptosis in hippocampus. INDO pre-treatment partly inhibited LPS-caused eNOS, iNOS and NOX4 up-regulation and further reduced 3-NT formation in hippocampus. INDO pre-treatment also partly ameliorated LPS-enhanced caspase-8 and Bax level. These data suggested that PGE2 was involved in NO and ROS production, and further influenced 3-NT formation in hippocampus. These alternations may finally induce apoptosis.Mitogen-activated protein kinases, (MAPKs) participate in several AD related molecular events. MAPKs signaling pathways are involved in protein regulations in early stage. The results shown that INDO inhibited LPS-induced ERK1/2 activation, yet the p38 was not affected by INDO intervention. These results suggested that PGE2 may regulated iNOS expression via ERK1/2 but not p38 signaling pathway.Overall, current data indicated that peripheral LPS induced PGE2 production in rats hippocampus. PGE2 may up-regulated NOSs and NOX4 expressions, which further lead to 3-NT formation and increased apoptosis tendency, suggesting an initiative role of PGE2 in inflammation induced pathological changes found in several neurodegenerative disorders. MAPKs signaling pathway are involved in these mentioned regulations.2. Involvement of TNF-αon BACE1 expression regulations in rats astrocytes, studies in vivo and in vitro.Present research was designed to investigate the potential roles of TNF-αand PGE2 on BACE1 expression regulations.BACE1 is a key enzyme participate in APP process and ultimately catalyze Aβformation. LPS (2 mg/kg, i.p.) injection led to BACE1 mRNA, protein and enzyme activity up-regulation as well as secreted APP (3 (sAPPβ) production. Double-staining by immunofluorescence indicated the up-regulated BACE1 were highly co-localized with glial fibrillary acidic protein (GFAP) positive astrocytes. These results indicated that LPS-induced inflammation induced astrocytes BACE1 up-regulation in rat hippocampus, suggesting inflammation may promote AD pathology by BACE1 up-regulation. In addition, these results also suggested astrocyte was the principle cell type involved in.To identify the role TNF-αplayed in LPS-induced BACE1 up-regulation. ETC was employed. As results shown, pre-treatment with ETC (5 mg/kg, suc.) significantly decreased LPS-induced BACE1 expression and sAPPβproduction in rats hippocampus. Localization trials indicated that BACE1 up-regulation can be observed in GFAP positive astrocytes but not in neurofilament positive neurons. These data indicated that blockage of TNF-αsignaling pathway was effective in reducing LPS-increased BACE1 expression and enzyme activity. To further explore identify the BACE1 regulation by TNF-αsignaling pathway, primary cultured rats astrocytes and neurons were incubated with TNF-αrespectively. As the results shown, after 24 h incubation with TNF-αin a range of 10-150 ng/ml, the BACE1 expression was up-regulated in astrocytes but not in neurons. The data obtained from both in vivo and in vitro suggested that TNF-αmay promote AD pathology by regulating BACE1 expression in astrocytes. TNF-αis a cytokine highly related with AD pathology.INDO (2 mg/kg, i.p.) pre-injection had no effects on LPS-induced BACE1 up-regulation, suggesting PGE2 may not involved in BACE1 regulations under inflammatory circumstance.Overall, current data collected from both in vivo and in vitro indicated that TNF-αwas involved in astrocytes BACE1 expression regulation, while PGE2 may not be a crucial factor in this process. These data suggested the important roles of TNF-αsignaling pathway and astrocytes in AD pathology. Summary1. Peripheral injection of LPS induced rats fever instantly, the mPGES-1 in brain cortex and hippocampus was up-rulated in an acute phase, as well as PGE2 content. The LTC4S is the only LTC4 synthase that found can be transiently up-rgulated, indicating that PGE2 may play a more important role in this model as an initial factor.2. Peripheral injection of LPS induced NOSs, NOX4 up-regulation, NO, ROS production,3-NT formation and apoptosis related proteins expression in hippocampus in a time sequence. These alternations can be partly inhibited by INDO administration, suggesting that PGE2 was involved in this process.3. In LPS-induced braininflammation in rats, TNF-αselectively regulated BACE1 expression in astrocytes both in vivo and in vitro, indicating astrocytes is an important in mediating inflammatory signals to early phase of AD pathology. |
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