Mechanism Of FLZ Controlling Neurodegenerative Disease By Modulation Of Inflammation And The Immunocompetence Of A Synthetic Muramyl Dipeptide Analogue MDP-C

The thesis is consist of two sections:"Mechanism of FLZ controlling neurodegenerative disease by modulation of inflammation" and "The immunocompetence of a synthetic muramyl dipeptide analogue MDP-C"Section 1.Mechanism of FLZ controlling neurodegenerative disease by modulation of inflammationCompound FLZ is a synthetic novel derivative of squamosamide.Previous studies in our laboratory demonstrated that FLZ not only had strong anti-oxidation, anti-apoptosis and neuroprotective effect,but also improved experimental learning and memory deficit and dyskinesia in animal models.This thesis first explored the ability of FLZ modulating inflammation and focused on mechanism studies controlling neurodegenerative disease.Part 1.FLZ improvement of learning and memory dysfunction that is caused by LPS-induced inflammation in hippocampusLipopolysaccharide(LPS) is the major component of the outer surface of gram-negative bacteria and be considered as a potent inflammatory stimulator. Experimental evidences indicated that intracerebroventricular(i.c.v.) injection of LPS to rodents induced learning and memory impairments as well as neurodegeneration in brain areas related to cognitive function.In the present study,we explored the effects of FLZ on the LPS i.c.v.injected mice models.In water maze test,we found that i.c.v.injection of 2.5μg LPS induced learning and memory impairment.FLZ(150 mg/kg,75mg/kg) significantly improved the learning and memory ability and inhibited the damage of hippocampus neurons as assessed by histopathology method.To further investigate the mechanism of FLZ in improving the learning and memory impairments induced by LPS, biochemistry and immunohistochemistry methods were used.The results revealed that FLZ decreased the levels of TNF-αand IL-1βand inhibited the activity of inducible nitric oxide synthase(iNOS) which were induced by LPS.The immunohistochemistry results showed that the increased activation of microglia and astrocyte and the over expression of iNOS,cyclooxygenase-2(Cox-2) andβ-Secretase(BACE1) by LPS were all significantly inhibited by FLZ.Aβproduction induced by LPS was decreased either. In summary,FLZ significantly inhibited the inflammatory reaction in hippocampus caused by i.c.v.injection of LPS and improved the learning and memory impairment and neuron damage.2.Inhibition effect of FLZ on the inflammatory reaction in macrophages and the mechanism of actionThe above results indicated that FLZ inhibited the inflammatory reaction in hippocampus caused by i.c.v.injection of LPS.To further study the mechanism of FLZ, the RAW264.7 macrophages were used to detect the effects of FLZ on the production of inflammatory mediators-induced by LPS.The results showed that FLZ dose-dependently inhibited the LPS induced production of nitric oxide(NO) as well as the expression of iNOS,cyclooxygenase 2(Cox-2) and tumor necrosis factor(TNF)-αat both mRNA and protein levels.Furthermore,the production of NO and secretion of TNF-αinduced by LPS or aggregated Aβ_1-40 in BV-2 microglias were also suppressed by FLZ.The anti-inflammatory mechanism of FLZ was investigated subsequently.In RAW264.7 cells, the LPS-induced DNA binding activation of Nuclear factor kappa-B(NF-κB) and activator protein 1(AP-1),the nuclear translocation of NF-κB p65,the degradation of inhibitoryκBαprotein(IκBα) and the phosphorylation of IκBα,IκB kinase(IKK)α/β, c-Jun NH₂-terminal kinase(JNK) and p38 mitogen-activated protein kinases(MAPKs) were all suppressed by FLZ.But the phosphorylation of extracellular signal-regulated kinase(ERK) was not impacted.Further study revealed that FLZ dose dependently inhibited the phosphorylation of transforming growth factor-β(TGF-β)-activated kinase 1(TAK1) which is the upstream signal of IKKα/β,JNK and p38 activation.In addition, reactive oxygen species(ROS) induced by LPS or H₂O₂ were suppressed by FLZ either. The results suggested that FLZ inhibited the production of inflammatory mediators at least partly via downregulation of TAK1-IKK-NF-κB and TAK1-JNK/p38MAPK/AP-1 pathways.In addition,the suppression of ROS was also one of the important mechanisms for the anti-inflammatory activity of FLZ.In summary,FLZ significantly inhibited the inflammatory reaction in hippocampus caused by i.c.v,injection of LPS and improved the learning and memory impairment and neuron damage.The production of inflammatory mediators induced by LPS was inhibited by FLZ at least partly via the downregulation of TAK1-IKK-NF-κB and TAK1-JNK/p38MAPK-AP-1 pathways and the inhibition of ROS.The protection against neurodegenerative diaseases of FLZ may be partially accounts for its anti-inflammation activity. Section 2.The immunocompetence of a synthetic muramyl dipeptide analogue MDP-CN-acetylmuramyl-L-alany-D-isoglutamin(muramyl dipeptide,MDP),which was identified as the minimal active component of mycobacterium cell wall,possesses extensive biological activities on host immune systems and improves the nonspecific resistance against infections and tumors.However,the characters of poor penetration of membranes and rapid elimination in vivo,as well as the induction of toxic responses, such as pyrogenicity,lethargy,anoresy and hypermetabolism limit the application of MDP.Therefore,structure modification and synthesis of MDP derivatives possess good activitie is extremely needed.MDP-C(N2-[R-Obenzyl-N-(acetylmuramyl) -L-alanyl -D-isoglutaminyl]-N6-trans-(m-nitrocinnamoyl)-L-lysine),a novel chemically modified MDP,has been reported to be an potential immunostimulator.In this study,we investigated the immunocompetence of MDP-C.First of all,we detected the effects of MDP-C on the growth and metastasis of tumors in a Lewis lung cancer mouse model.The results showed that MDP-C has no inhibitory activity on both the primarily tumor and pulmonary metastasis.Under tested condition,at the dose of 1.25 mg/kg and 2.5mg/kg,MDP-C combined with low dose isofosfamide(15 mg/kg) decreased the numbers of pulmonary metastasis nodes.But the data have no statistical significance compared with control group.In the experiment of ELISPOT,MDP-C slightly augmented the TRP-2_180-188 peptide-specific cytotoxic T lymphocytes(CTLs) reaction in spleen cells of C57BL/6J mice immunized with the TRP-2_180-188 peptide and MDP-C,but have no statistical significance compared with TRP-2_180-188 peptide group.Dendritic cells(DCs) are highly specialized antigen-presenting cells(APC) that play a central role in the initiation of both innate and adaptive immune responses.The effects of MDP-C on the differentiation and activation of human monocyte derived dendritic cells(MoDCs) were investigated.The combined action of GM-CSF and IL-4 could direct monocytes to differentiate into DCs displaying features of immature DC(iDCs).After cultured with various concentration of MDP-C for 24h,the surface molecules human leucocyte antigen-DR(HLA-DR),CD80,CD86 and CD83 were detected by flow cytometry.The allogeneic mixed lymphocyte reaction and the levels of IL-6 and IL-12(p40+70) were investigated with ATP bioluminescent assay kit and ELISA kit, respectively.The results showed that MDP-C up-regulated the expression of HLA-DR, CD80,CD86 and CD83,as well as the allogeneic mixed lymphocyte reaction.But the secretion of IL-6 and IL-12(p40+70) were not affected.While the positive control compound romurtide induced the expression of surface molecules and cytokines strongly. Further study revealed that MDP-C has no effect on the secretion of TNF-αin both RAW264.7 and mouse peritoneal macrophages.In summary,MDP-C has no obvious inhibitory effect on the growth and metastasize of tumors in the Lewis lung cancer mouse model and has no obvious adjuvant activity on the TRP-2_180-188 peptide induced cytotoxic T lymphocytes(CTLs) activition under tested condition.MDP-C increased the expression of surface molecules and enhanced the antigen presenting activity of MoDCs,but has no effect on the cytokine expression in both MoDCs and macrophages.The results indicated that MDP-C possesses immunocompetence to some extent.The mechanism of action needs further investigation.