| Background and Objectives: Epilepsy is a complex brain disorder characterized predominantly by abnormal excessive or synchronous neuronal activity.With about 70 million people affected globally,epilepsy has not only became one of the most common and serious disease but also heavily burdened both families and the society.The pathogenesis of epilepsy is complicated and it is generally believed that there is excessive excitement of neurons during its development,that is,the imbalance of excitatory glutamatergic transmission and inhibitory GABAergic transmission.At present,the choice of treatment strategies for epilepsy is mainly based on its pathogenesis,but there is still at least one third of patients who do not adequately respond to therapies.Therefore,in-depth research on the pathogenesis of epilepsy is particularly important.Recently,numerous studies have confirmed immune abnormalities during the onset of epilepsy and increasing studies have focused on the immune and inflammatory component in this disease.As one of the most important immunological factors,microglia have also become the focus of research.As the major resident innate immune cells in brain,microglia play an important role in maintaining normal nerve function and participating in the pathophysiology of diseases.In the physiological state,microglia actively survey the surrounding environment,regulate neurogenesis or oligodendrogenesis,phagocytose apoptotic neurons and promote neuronal survival,as well as control synaptic density and connectivity by prune synapses.However,in an over-activated state,microglia may produce and release excessive neurotoxic substances such as nitric oxide,which may cause an unmanageable level of oxidative stress,thereby aggravating the death or injury of nerve cells.In epilepsy brain tissue,microglia can be either pro-epileptic or anti-epileptic,which may be related to its polymorphism.Based on this theory,the activation of metabotropic glutamate receptors may switchmicroglia to more complex phenotypes.More attention should be paid to the role of microglial cells in different stages of epilepsy.At present,there are few studies focus on the role of resting,pro-inflammatory and anti-inflammatory microglia play on the excitability of neurons in response to metabolic glutamate receptor agonist.Based on the establishment of microglia models with different activation states,our research aims to study the changes and the mechanism of microglial functional state,and focuse on the search for cell survival and pathology pathways related to epilepsy and verify its role,exploring the possibility of regulating microglial activation and inflammatory response as a strategy for epilepsy treatment.Materials and Methods:(1)N13 microglia cell line was used for cell culture.Lipopolysaccharide(LPS),interleukin-4(IL-4)/ interleukin-10(IL-10)/ transforming growth factor ?(TGF-?),and metabolic glutamate receptor agonists such as DHPG,LY 354740,and L-AP4 were used to establish model of microglia in the rest state,pro-inflammatory state and anti-inflammatory state with or without m Glu Rs activated.Fluorometric assay and ELISA were used to detect the production of nitric oxide(NO)and cytokines such as interleukin-1?(IL-1?)?interleukin-6(IL-6)?IL-4 and Tumor necrosis factor alpha(TNF-?).(2)N13 microglia cell line was used for cell culture.LPS and metabolic glutamate receptor agonists such as DHPG and L-AP4 were used to establish model of microglia in the rest state and pro-inflammatory state with or without m Glu Rs activated.Western Blot was used to screening the key molecules in microglial inflammation-relevant pathways and glutamate signaling pathways.q PCR was used to detect the expression of Long non-coding RNAs.(3)C1300 neuron cell line was used for cell culture.Microglial supernatants of resting,pro-inflammatory and anti-inflammatory states with or without m Glu Rs activated were used to stimulate neurons to establish different neuronal model.Neuronal activity and damage were assessed by measuring CCK8,LDH levels and glutamate release.Western Blot was used to detect the protein expression levels of n-methyl-d-aspartate receptor(NMDAR),calmodulin-dependent protein kinase II(Ca MK II),inducible nitricoxidesynthase(i NOS)which related to neuronal excitability.(4)The data were presented as mean value ± SD.The statistical program for social sciences(SPSS)22.0(SPSS,USA)was used to analyze all the data.One-way analysis of variance(ANOVA)and Tukey?s post hoc test were used to compare values among groups.For all tests,the level of significance was set to P< 0.05 and it was shown in the figures(*P< 0.05).Results:(1)m GLu R I activated microglia significantly attenuated the release of IL-1? and IL-6 but increased the release of IL-4 and TNF-? in inflammatory conditions.m GLu R II activated microglia significantly increased the release of IL-1? in inflammatory conditions.m GLu R III activated microglia significantly attenuated the release of IL-6 in inflammatory conditions.(2)m GLu R I activated microglia significantly attenuated the release of TNF-? but increased the release of IL-4 and IL-6 in anti-inflammatory conditions.m GLu R II activated microglia significantly attenuated the release of TNF-? but increased the release of IL-4 in anti-inflammatory conditions.m GLu R III activated microglia significantly attenuated the release of IL-1? but increased the release of IL-4 and IL-6 in anti-inflammatory conditions.(3)Both DHPG and L-AP4 were able to inhibited the NF-?b p65 expressions in LPS-activated microglia,thereby inhibited the activation of NF-?b signaling pathways.PKA signaling pathways were activated after adding DHPG and L-AP4 in both resting and LPS-activated microglia.Both DHPG and L-AP4 significantly reduced LPS-enhanced p44/42 MAPK levels in activated microglia.(4)DHPG significantly enhanced PI3 Kinase and GSK-3? expressions in LPS-activated microglia.However,there was no significant difference regarding the activation levels in LPS-treated microglia after L-AP4 stimulated.L-AP4 significantly reduced GSK-3? expressions in LPS-activated microglia.(5)DHPG significantly enhanced linc RNA-COX2 and linc RNA-EPS expressions in LPS-activated microglia.L-AP4 significantly reduced both linc RNA-COX2 and linc RNA-EPS levels in LPS-activated microglia.(6)Microglial supernatant significantly increased the neuronal viability as well asreduce neuron death and damaged in inflammatory conditions after m GLu R I activated and anti-inflammatory conditions m GLu R III activate.(7)Microglial supernatant significantly increased the release of glutamate in physiological conditions.Microglial supernatant significantly attenuated glutamate release in inflammatory conditions after m GLu R I activated and anti-inflammatory conditions m GLu R III activated.(8)Microglial supernatant significantly inhibited the activation level of NR1?NR2A and NR2 B of neuron in LPS-activated microglia after m Glu R I or m GLu R III activated,as well as increased the activation level of Ca MK II of neuron.Microglial supernatant significantly inhibited the activation level of NR2 B of neuron in LPS-activated microglia after m GLu R II activated.Microglial supernatant significantly increased the activation level of NR2 A and NR2 B of neuron in IL-4/IL-10/TGF-?-activated microglia after m Glu R I activated.Microglial supernatant significantly inhibited the activation level of NR2 A of neuron in IL-4/IL-10/TGF-?-activated microglia after m Glu R II activated.Microglial supernatant significantly inhibited the activation level of NR1 and NR2 A of neuron in IL-4/IL-10/TGF-?-activated microglia after m Glu R III activated.Microglial supernatant significantly increased the activation level of Ca MK II of neuron in IL-4/IL-10/TGF-?-activated microglia after m Glu R I or m GLu R II activated.(9)Microglial supernatant significantly increased the release of NO in inflammatory conditions.Microglial supernatant significantly increased the release of NO in inflammatory conditions after m Glu R I activated.Microglial supernatant significantly inhibited the i NOS expressions of neuron in LPS-activated microglia after m Glu R I activated.There was no significant difference regarding the release of NO in IL-4/IL-10/TGF-? treated microglia after DHPG?LY 354740 and L-AP4 stimulated.Microglial supernatant significantly inhibited the neuronal i NOS expressions in IL-4/IL-10/TGF-? stimulated microglia after m Glu R I or m GLu R III activated.Conclusions:(1)The activation of m Glu R I tended to restrain existing inflammations by either downregulating pro-inflammatory cytokine secretions or upregulating anti-inflammatory cytokine release in LPS-stimulated microglia.The activation of m Glu R III had the capacityto transform LPS-activated microglia to resting states.However,the activation of m Glu R III in IL-4/IL-10/TGF-? stimulated microglia tended to maintaine its activated state.(2)The polymorphisms of m Glu R agonists for the regulation of microglia in different states closely related to the antagonistic relationship between the MAPK – ERK-Gsk3? pathway and the PI3 K – Akt-Gsk3? pathway.(3)m GLu R I activated microglia in the inflammatory state and m GLu R III activated microglia in the anti-inflammatory state increased the vitality of neurons,reduced neuronal death and damage,reduced the release of excitatory amino acids,and also down-regulated the expression of excitability related pathway protein.In this situation,microglia had a potential effect of antiepileptic and neuronal protection. |
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