The Effect Of Nicotine On The Activation And Resultant Death Of Microglia Induced By Lipopolysaccharide

ObjectiveMany researchs found that the overactivation of microglia may cause severe brain tissue damage in various neurodegenerative diseases.Inversely,repressed the overactivation of microglial cells would benefit to recover these diseases in CNS.Nicotine(NIC),is the major alkaloid of tobacco,which smoking has been the most popular method of nicotine intaking. For a long time,nicotine had been simply regarded to be bad effect for the health and be a primary risk factor in the process of lung cancer,abortion,addiction danger,cardiovascular disorders,pulmonary disease and toxic damage in nervous system,et al.On the other hand, recent researchs reported that nicotine also produced lots of diverse effects on the CNS by provided with different dose and time,some of them may be considered to be beneficial, such as mood elevation,attention concentration,learning and memory enhancement. Furthermore,nicotine as a potential method for the therapy of Alzheimer's disease,Parkinson's disease,Ulcerative colitis,Septicemia,Ulceratiue stomatitis and so on has been investigated.More and more proofs indicated that nicotine possessed a lot of good effect for the body.Nicotine had been proved to can modulate the innate and adaptive immune responses and inhibit the inflammatory states.Cytokines(including antiinflammatory and proinflammatory factor)play a crucial role in regulating the inflammatory reaction. Nicotine could suppress the gene expression of proinflammatory cytokines and modulate cytokines release through binding endogenousα7nACHRs in inflammation.In addition, cholinergic antiinflammatory mechanisms also play an important role in inflammatory progress.At the same time,several nicotinic acetylcholinergic receptor subtypes(nACHRs) had been identified in B and T lymphocytes,monocytes,macrophages,microglia,dendritic cells and endothelial cells,et al.Now,the antiinflammatory activity of nicotine throughα7nACHRs by activating the cholinergic antiinflammatory pathways was obvious more and more,but the detailed mechanism kept obscure.We made the following experiments to observe the effect of nicotine on the activation and resultant death of microglia induced by LPS in vivo and in vitro,which was helpful to explore and understand the nicotinic antiinflammatory mechanisms in inflammation of CNS.Methods(一)Experiments in vivo1,In vivo,male,healthy experimental animals were randomly divided into CON,NIC,LPS,NIC+LPS group.The animal model that was exposed to chronic nicotine treatment by intraperitoneal injection was established according the experimental rule, and LPS was provided intraperitoneally to induce the activity of microglia.2,The CD11b-positive microglia of every group were compared among Cerebral cortex,Hippocampal and Substantia ngra(SN)through immunohistochemistry staining.(二)Experiments in invtro1,The culture of BV2 cells and divided into CON,NIC,LPS,NIC+LPS group.2,CCK-8 assay cell activity of BV2 cells in the different experimental group.3,Nitric Oxide Assay Kit assay NO production of BV2 cells in the different experimental group.4,RT-PCR assay the expression of iNOS mRNA,IL-1βmRNA,IL-6 mRNA,TNF-αmRNA,COX-2 mRNA,IRF-1 mRNA,Caspase-11 mRNA of BV2 cells in the different experimental group.5,Western-Blot assay the protein expression of Caspase-3 and P-I-κB of BV2 cells in the different experimental group.Results1,The CD11b-positive microglia were dyed low brown with small bacilliform cell bodies,ramified morphology,thin and long protuberance in CON and NIC group;In LPS group,the CD11b-positive microglia were dyed chocolate brown with more big roundish cell bodies,thicker and shorter processes;Most CD11b-positive microglia were dyed brown with bacilliform cell bodies,ramified morphology and few cells were dyed deep brown with more big roundish cell bodies,thicker and shorter protuberance in NIC+LPS group.2,Cell viability was declined with the time prolong.BV2 cells growed well with ramified morphology in CON and NIC group;Most cells were suspended and decomposed in LPS group;AICD was attenuated in NIC+LPS group.3,NO release was increased with the time prolong.At the different time point, LPS induced markly NO release and NO production was reduced in NIC+LPS group.4,LPS induced immensely the mRNA expression of proinflammatory cytokines including iNOS,IL-1β,IL-6,TNF-α,COX-2,IRF-1,Caspase-11 of BV2 cells;In NIC+LPS group,the mRNA expression of cytokines were suppressed.5,LPS induced tremendously the protein expression of P-I-κB and activated Caspase-3(20KD);In NIC+LPS group,the protein expression were decreased.ConclusionNicotine pretreatment can repress the activation and activation-induced cell death of microglial cells induced by LPS,which suggests that it may play a neuroprotective role on inflammatory reaction of brain.