The Effects And Mechanisms Of Oxytocin In Lipopolysaccharide-induced Microglial Activation

Background:Neuroinflammation mediated by glial activation is identified as a common character during progression of many neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD). Microglia, the resident immune cells of the central nervous system (CNS), is considered to play a key role in regulating neurotoxicity mediated by inflammatory response. Microglial activation can be induced by lipopolysaccharide (LPS), interferon (IFN)-γ or β-amyloid and results in overproduction of inflammatory cytokines. These inflammatory mediators, such as tumor necrosis factor-a (TNF-a), interleukin (IL)-6, IL-1β, glutamate, nitric oxide and reactive oxygen species, can collectively lead to neuronal damage, resulting in the progress of neurodegenerative diseases. During this process, ramified resting microglia undergo morphological transformations including deramification, process shortening and thickening, and finally development into its activated amoeboid form. Therefore, anti-inflammatory treatment via inhibition of microglial activation is regarded as a promising strategy for preventing neurodegenerative diseases in the clinic.Oxytocin (OT), a nonapeptide produced in the paraventricular and supraoptic nuclei of the hypothalamus, has a wide range of effects in the body. OT exerts its effects via G-protein-coupled receptors, which are expressed abundantly in the central and peripheral nervous systems. Recent data indicate that OT may also have anti-inflammatory and anti-oxidant properties, and regulates the immune and anti-inflammatory response. Exogenous OT administration alleviates tissue damage in a variety of animal models of injury. Furthermore, co-administration of an OT receptor antagonist blocks the protective effects of OT during cardiac ischemia or cerebral ischemia in rats. The protective actions of OT in these models may be associated with decreased levels of circulating pro-inflammatory cytokines and decreased neutrophil infiltration to the site of injury. Moreover, OT inhibits LPS-stimulated pro-inflammatory cytokines secretion from macrophages and endothelial cells.However, little information is available about the effects of OT on neuroinflammation and its underlying molecular mechanisms. Therefore, we aimed to investigate the anti-inflammatory effects of OT on LPS-stimulated microglial activation, and its therapeutic effects on the early stage of neuroinflammation induced by systemic LPS treatment in mice.Objective:We established the inflammation models in vitro and in vivo by using LPS. Then we investigated the anti-inflammatory effects of OT on LPS-stimulated microglial activation.We investigated its therapeutic effects on the early stage of neuroinflammation induced by systemic LPS treatment in mice.Methods:Using the BV-2 microglial cell line and primary microglia.BV-2 cells and primary microglia were per-treated with OT (0.1,1 and 10μM) for 2h followed by LPS treatment (500 ng/ml), microglial activation and pro-inflammatory mediators were measured by Western blot, RT-PCR and immunofluorescence. The MAPK and NF-κB pathway proteins were assessed by Western blot. The intracellular calcium concentration ([Ca2+]i) was determined using Fluo2-/AM assay. Intranasal application of OT was per-treated in BALB/C mice (adult male) followed by injected intraperitoneally with LPS (5 mg/kg). The effect of OT on LPS-induced microglial activation and pro-inflammatory mediators were measured by Western blot, RT-PCR and immunofluorescence in vivo.Results:OTR expression was examined in BV-2 cells and primary microglia. MTT results demonstrated that either treatment of OT alone or with LPS had no effect on the cell viability of cells in the performed concentrations. Microglial cells are activated in response to different stimuli, which lead to morphological and functional changes.We found that OT pre-treatment significantly inhibited LPS-induced microglial activation and reduced subsequent release of pro-inflammatory factors. In addition, OT inhibited phosphorylation of ERK and p38 but not JNK MAPK in LPS-induced microglia. Furthermore, OT remarkably reduced the elevation of [Ca2+]i in LPS-stimulated BV-2 cells. Further a systemic LPS treatment acute inflammation murine brain model was used to study the suppressive effects of OT against neuroinflammation in vivo. We found that pre-treatment with OT demonstrated marked attenuation of microglial activation and pro-inflammatory factors levels.Conclusions:1. OT significantly attenuates overactivation of microglial cells.2. OT reduces expression levels of pro-inflammatory mediators and cytokines via inactivation of ERK/p38 MAPK signaling pathways.3. OT would be a potential therapeutic agent for alleviating neuroinflammatory diseases accompanied by activated microglia.