| Recent studies indicated that inflammation play an important role in the pathogenesisof diseases with high morbidity and mortality in the spectrum of diseases or chronicdiseases with heavy burden, which like tumor, cardiovascular and cerebrovascular diseases,metabolic diseases, neurodegenerative diseases, etc. The regulation of inflammation iscomplex and accurate. The nervous system, endocrine system and immune system areinvolved in the regulation. The neuro-endocrine-immune network is critical in theregulation of inflammation and homeostasis.Neuropeptide Y (NPY) is a36-amino acid peptide neurotransmitter first found in thebrain of pigs by Tatemoto and Mutt from Karolinska college in1982. It is widelyexpressed in the central or peripheral nervous system. NPY has been associated with anumber of physiologic processes in the brain, including the regulation of energy balance,memory and learning, endocrine and reproduction. In addition, The role of NPY in immuneregulation and the pathogenesis of autoimmune diseases is gradually disclosed. Inperiphery, sympathetic nerves are the capital source of NPY. It is found that peripherysympathetic nerves govern immune organs in human body. There is even "synapse-like"structure formed between the endings of sympathetic nerves and macrophages. Thisphenomenon has provided anatomical bases of NPY's role in modulation of inflammationand functions of macrophages.Macrophage is an important component of innate immune system. It is derived fromperiphery blood monocyte and widely located in all tissues of body. The activatedmacrophages could secreted proinflammatory cytokines such as IL-1β, IL-6, TNF-α orHMGB1and inflammatory mediators such as leukotrienes, prostaglandins, elastases,muramidases or urokinases. Both of proinflammatory cytokines and inflammatorymediators could aggravate the inflammation. To characterize the role of NPY in themodulation of macrophage's secretion of proinflammatory cytokines and inflammatorymediators is an urgent problem needed to be solved in neuroimmunomodulation.Systems biology is described as a biology-based inter-disciplinary study field thatfocuses on complex interactions in biological systems.Metabolomics is the scientific study of chemical processes involving metabolites.Specifically, metabolomics is the "systematic study of the unique chemical fingerprints thatspecific cellular processes leave behind", the study of their small-molecule metabolite profiles. The metabolome represents the collection of all metabolites in a biological cell,tissue, organ or organism, which are the end products of cellular processes. Thus, whilemRNA gene expression data and proteomic analyses do not tell the whole story of whatmight be happening in a cell, metabolic profiling can give an instantaneous snapshot of thephysiology of that cell. By using metabolomics, we could get the changes of metabolomeof supernatant of macrophages treated with NPY and LPS compared with LPS-only treatedmacrophages. Proteins are central to our understanding of cellular function and diseaseprocesses. Proteomics in general deals with the large-scale determination of gene andcellular function directly at the proteome level. By using bioinformatic tools and documentwork, we might find important molecules in NPY regulation of inflammation.We carried out our work on mouse macrophage. First, LPS was adopted to elicitinflammation in macrophage. Major proinflammatory cytokines were detected to accessNPY regulation on inflammation. On the other hand, Metabolomics were performed toscreen small-molecule metabolites. Then, a novel high-throughput comparative proteomicapproach based on2D-nano-LC-MS/MS was applied to simultaneously evaluate thederegulated proteins involved in the response of NPY treatment in RAW264.7cells. Theproteomes were divided in two cellular compartments (nucleus and cytoplasm). Followingthe results of proteomics, we supposed possible mechanisms in NPY regulation ofinflammation and doing some work to confirm our hypothesis. The detailed results werepresented as follows:1. In the present study, LPS-induced inflammation in macrophage was established. Wefound that NPY could suppress TNF-α, IL-1β and IL-6secretion. Interestingly, weobserved that NPY could significantly enhanced HMGB1secretion, which is an activeHMGB1release. Then, we found NPY could elevate HMGB1mRNA levels faster thanLPS. By using metabolomics, we learned the changes of metabolome of supernatant ofmacrophages treated with NPY and LPS compared with LPS-only treated macrophages.After validations of these molecules, we demonstrated that lipoxin A4and prostaglandinB2were down-regulated for0.53folds or0.54folds, respectively.2. Proteomics assay was employed to explore possible mechanisms underlying NPYregulation of inflammation.2D-nano-LC-MS/MS was applied to simultaneously evaluatethe deregulated proteins involved in the response of10-8mol/L NPY treatment inRAW264.7cells. The nucleus and cytoplasm part of the proteome were separated in theassay. A total of435proteins were differentially expressed. After using bioinformatic tools to analyze the data, we found that the function of these proteins covers diverse facetincludes energy metabolism, cytoskeleton, nucleic acid metabolism and inflammatoryfunction. We discussed several molecules and claimed that a joint molecule, named β-arrestin2, might take part in the modulation of inflammation by its transition from cytosolto the nucleus. The current study also indicated proteomics assay based on2D-nano-LC-MS/MS combined with bioinformatics is a useful tool for study of molecularmechanism.3. NPY exerts its action through binding to the NPY receptors which are members ofthe G protein-coupled receptor (GPCR) superfamily. Y1receptors are the main functionreceptor expressed in macrophages. Molecular mechanisms of NPY function is reportedrarely so far. We combined the first and second part results together and exhibited that β-arrestin2transferred from the cytoplasm to the nucleus. Meanwhile, p65transitioninduced by LPS stimulus was suppressed when NPY was added with LPS, which meansthe transcription activity of NF-κB was inhibited.
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