| Microglia are mononuclear macrophages that reside in the central nervous system(CNS)and account for about 10-20% of the total glial cells of the brain.Microglia is the first line of defense against intracerebral immune stimuli.The functions of microglia are mainly phagocytosis,antigen presentation,and production of cytokines.Microglia are generally divided into "resting state" and "activated state".However,the "resting state" of microglia is not a true functional quiescence.Two-photon microscopy finds that even the "resting state" of microglia is also constantly engulfing the surrounding body fluids to ensure the balance of the immune environment and plays the key role in immune surveillance.Once encountered with foreign substances or noxious factors,microglia quickly turn into the “activated state” as well as synthesize and release large amounts of inflammatory and neurotoxic substances,including tumor necrosis factor ?(TNF-?),interleukin-1?(IL-1?),interleukin-6(IL-6),prostaglandin E2(PGE2),nitric oxide(NO),and reactive oxygen species(ROS).These substances are mainly produced to regulate CNS innate immunity and initiate central inflammation.The central role of central inflammation is to protect the CNS.However,uncontrolled and prolonged central inflammation may be detrimental and eventually produce cell damage,which is closely related the occurrence of neurodegenerative diseases.Therefore,activation of microglia is an important research target for the pathological process of neurodegenerative diseases.Histamine is well known for its important role in allergic reactions and gastrointestinal inflammation.CNS also contains histaminergic neurons,which are closely related to sleep,eating,attention,and cognition.Histamine is synthesized by histidine decarboxylase,which acts through four histamine subtype receptors,such as H1 R,H2R,H3 R,and H4 R.These four receptors are G protein-coupled receptors.H1 R is closely related to allergic reactions,and its activation can lead to capillary dilation,increased permeability,and smooth muscle spasm.H2 R is related to secretion of gastric acid and bronchial gland secretion by gastric parietal cells.H3 R is distributed in central and peripheral nerve endings,regulating the synthesis and release of histamine and other neurotransmitters.The H4 R is the latest discovery of histamine receptors,which can mediate the release of cytokines from immune cells such as T cells.Our previous study found that microglia also contained four histamine subtype receptors,and histamine could activate microglia through H1R/H4R-PI3K/AKT/MAPK-NF-?B signaling.However,the role of microglia histamine receptors in central inflammation remains unclear.Therefore,this study will investigate effects of microglia histamine receptors in LPS-induced central inflammation both in vitro and in vivo.Part ? Histamine Induces Upregulated Expression of Histamine Receptors and Increases Release of Inflammatory Mediators from Microglia in vitroObjective: To investigate effects of histamine receptors on the occurrence of LPS-induced microglia activation in vitro.Method: 1.By using flow cytometry,four histamine receptors on microglia induced by 10ng/ml of LPS were detected.2.First,microglia was exposed to various concentration of histamine(0.001,0.01,0.1,and 1 ?g/m L)plus LPS for 24 h.ELISA was used to detect production of TNF-? in culture medium.Second,microglia were exposed to 1 ?g/m L of histamine and LPS for 1.5,3,6,12,and 24 h.Then,ELISA was used to detect production of TNF-? in culture medium.3.Thirty minutes ahead of microglia exposed to LPS,microglia were exposed to various concentration of antagonists of four histamine receptors(0.1,1,10,and 100 ?M)for 24 h.ELISA was used to detect production of TNF-? in culture medium.Microglia were preincubated with 10 ?M of antagonists of four histamine receptors after they were exposed to histamine and LPS for 24 h,.ELISA was used to detect production of TNF-? in culture medium.4.Thirty minutes ahead of microglia exposed to LPS,microglia were exposed to various concentration of agonists of four histamine receptors(0.1,1,10,and 100 ?M)for 24 h.ELISA was used to detect production of TNF-? in culture medium.Results: 1.LPS(10 ng/m L)induced the increasing H2 R and H3 R,but not H1 R or H4 R expression of microglial.2.LPS(10 ng/m L)could stimulate the production of TNF-? released from microglia at 24 hours,and histamine could also induce the release of TNF-? in microglia dose-dependently.However,histamine(0.1 and 1?g/m L)could partially inhibit theproduction of TNF-? induced by LPS when both of them work together on microglia.3.Antagonists of H1 R and H4 R could partially inhibit LPS-induced release of TNF-?.However,antagonists of H2 R and H3 R have a synergistic effect on LPS-induced microglia activation and partially block the inhibitory effect of histamine on LPS-induced TNF-? production.4.Agonist of H1R(1-100 ?M)had a synergistic effect on LPS-induced microglia activation at 24 hours,which could be blocked by H1 R antagonist.Agonists of H2 R and H3R(1-100?M)could partially inhibit LPS-induced release of TNF-?,and also could be blocked by a corresponding receptor antagonist.Agonist of H4 R has no significant effect on LPS-induced release of TNF-?.Conclusions: This study demonstrated that Primary microglia stimulated by LPS in vitro experiments,H1 R and H4 R on microglia have pro-inflammatory effects,H2 R and H3 R have anti-inflammatory effects.Histamine partially inhibits LPS-induced TNF-? production by activating H2 R and H3 R.Part ? Effect of Histamine Receptors on LPS-induced Acute Neuroinflammation in vivoObjective: To investigate effects of histamine receptors on LPS-induced acute neuroinflammation in vivo.Method: 1.LPS(10 ?g/kg)was intraperitoneally injected and then the rats were sacrificed at 0.5,8,and 24 h later.The contents of histamine in the hypothalamus and cortex were detected by HPLC.2.Intraperitoneal injection of LPS was combined with or without 80 ?g of histamine injected with lateral ventricle localization.Then the rats were sacrificed at 8 and 24 h later.Immunohistochemistry was used to detect activation of microglia in the cortex.3.Intraperitoneal injection of LPS was combined with or without injection of histamine into the lateral ventricle.Then the rats were sacrificed at 0.5,8,24,and 72 h.ELISA was used to detect the contents of TNF-?,IL-1?,and IL-10 in the cortex.4.Thirty minutes ahead of intraperitoneal injection of LPS,agonists or antagonists of histamine receptors were injected in the lateral ventricle.Eight hours later,the rats were sacrificed.ELISA was used to detect the contents of TNF-?,IL-1?,and IL-10 in the cortex.Results: 1.The level of histamine increased significantly in hypothalamus at 30 minutes after intraperitoneal injection of LPS(10?g/kg),and got the peak level at 8 and 24 h.However,the histamine level of cerebral cortex had significantly difference at 8 and 24 h,but not 30 min after LPS intraperitoneal injection.2.Intraperitoneal injection of LPS(10?g/kg)could induce microglia activation in cerebral cortex significantly at 8 and 24 h,while the intracerebral coadministration of histamine(80?g)could partially inhibit this phenomenon.3.Agonist of H1 R cooperated with LPS to promote the release of pro-inflammatory cytokines,but its antagonist had the opposite effect.Agonists of H2 R and H3 R inhibited LPS-induced neuroinflammation and increased the expression of IL-10,but their antagonists had the opposite effects.Conclusions: This study demonstrated that in vivo experiments,H2 R and H3 R play an anti-inflammatory role in LPS-induced neuroinflammation of Wistar rats,H1 R plays a pro-inflammatory role,whereas H4 R does not have effect. |
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