The Mechanism Of Chronic Cold Exposure Up-regulated GABA Inducing Microglia Activation In Hippocampus Of Mice

Cold stress is one of the most common stress responses in northern China.Chronic cold stress not only leads to a disruption of internal environment homeostasis,affects the immune function of animals,but also causes adverse impacts to the neuroendocrine system.Previous studies in our laboratory have found that chronic cold stress leads to disorders of neurotransmitter metabolism in the hippocampus of mice,especially ?-aminobutyric acid(GABA),the number of mature neurons in the hippocampus also declined significantly,accompanied by the activation of microglia,suggesting the occurrence of neuroinflammation.However,the specific mechanism is still to be further studied.Then,what is the mechanism of microglia activation under cold exposure? Is this neuroinflammation related to the metabolic disorder of neurotransmitters? Therefore,to investigate the response of hippocampus to cold exposure and clarify the mechanism of microglia activation induced by cold exposure in the hippocampus of mice,which are of great significance for revealing the pathogenic mechanism of cold stress and proposing protective measures for stress injury.In order to explore the effects of chronic cold exposure on the internal environment of mice and the hippocampus,5-week-old male C57BL/6 mice were randomly divided into a cold exposure group(CE)and a room temperature group(RT).The CE group was transferred into a4°C artificial intelligence climate room for 3 h each day,7 days later,blood-gas analysis was combined with ELISA to detect the acid-base balance of mice,neurotransmitter and the release of stress hormone.The results showed that after cold exposure,the arterial PCO2(P<0.05),t CO2(P<0.01),p H value(P<0.01)and K+(P<0.05)all showed significant changes,and the levels of GABA(P<0.01)and corticosterone(CORT)(P<0.0001)in the serum of the mice increased significant,indicating that chronic cold exposure induced the disturbance of the homeostasis in mice.The impact of chronic cold exposure on neurons of hippocampus in mice was evaluated by immunohistochemistry,immunofluorescence,Nissl staining,western blot and other methods.Then detection the state of glial cells,the expression of neuronal marker microtubule-associated protein 2(MAP-2),key proteins of nucleotide-binding domain(NOD)-like receptor(NLR)protein 3(NLRP3)inflammasome and nuclear factor kappa-B(NF-?B)signaling pathway.Result showed that,compared with the RT group,the expression of the heat shock protein 70(Hsp70)in the hippocampus of CE group was significantly increased(P<0.01),and the expression of brain-derived neurotrophic factor(BDNF)(P<0.01),C-FOS(P<0.0001),MAP-2,the number of Nissl bodies were all significantly reduced.Besides,astrocytes in the hippocampus was increased in the CE group,microglia were activated,the expression of tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)also increased significantly(P<0.01).Additionally,the expression of GABA receptors BABAB1(P<0.001)and GABAB2(P<0.01),NLRP3(P<0.001)and cysteine-requiring aspartate protease-1(Caspase-1)(P<0.05),which are key proteins of NLRP3 inflammasome signaling pathway,were significantly increased.The expression of deacetylase sirtuin1(SIRT1)(P<0.05)was significantly decreased,and the acetylation level of p65(P<0.01)was significantly increased following cold exposure.Indicating that chronic cold exposure led to the disturbance of the homeostasis in the mice.Neurons were damaged,microglia were activated,and the NLRP3 inflammasome and NF-?B signaling pathway were activated.In order to explore the effects of GABA on NLRP3 inflammasome and NF-?B signaling pathway in BV2 cells,different concentrations of GABA were used to treat BV2 cells,q PCR was combined with CCK-8 cell viability assay to screen the activation concentration of microglia.The results showed that when treated with GABA in 200 ?M,the expression of IL-1? was significantly increased(P<0.01),in the meanwhile there was no significant difference in cell viability(P>0.5).Besides,ionized calcium binding adaptor molecule-1(IBA-1)and CD11 b positive cells,the markers of microglial activation,and proinflammatory cytokines(P<0.01)were significant increased,indicating that microglia were activated.Then the activation of inflammation-related signaling pathways was detected by immunofluorescence,western blot,while the promoters' activity of key protein was detected by dual luciferase reporter gene assays.Result showed that,the expression of GABAB1(P<0.001),GABAB2(P<0.001),NLRP3(P<0.001)and Cleaved-caspase-1(P<0.0001)were significantly increased.Indicating that the NLRP3 inflammasome signaling pathway was activated.Additionally,the expression of SIRT1(P<0.01)was significantly decreased,and acetylation level of p65(P<0.001)was significantly increased,and the levels of acetylation of p65(P<0.001)and histone H3(P<0.0001)in the nucleus were significantly increased,and the activity of the p65 promoter is significantly enhanced(P<0.05).Showing that NF-?B signaling pathway was activated.In order to verify the relationship between GABA-mediated BV2 cell activation and NLRP3 inflammasome and NF-?B signaling pathway,BV2 cells were treated with different concentrations of NLRP3 inflammasome signaling pathway inhibitor MCC950 and NF-?B signaling pathway inhibitor BAY 11-7082,after 6 h,treated with GABA(200 ?M)for 3 h.Screening the inhibitory concentration of inhibitors by q PCR and CCK-8 cell viability assay,then the effects of the two inhibitors on NLRP3 inflammasome and the NF-?B signaling pathway was assessed by immunofluorescence,western blot,and dual luciferase reporter gene assays.The results showed that MCC950(100 ?M)and BAY(10 ?M)can effectively inhibit the activation of NLRP3 inflammasome and NF-?B signaling pathway.Finally,evaluate the effects of inhibitor on the activation of BV2 cells induced by GABA.It was showed that both MCC950 and BAY treatment can significantly inhibit the increase of IL-1?(P<0.001)and IBA-1(P<0.0001,P<0.01)induced by GABA in BV2 cells.The result indicates that the NLRP3 inflammasome and NF-?B signaling pathway mediate BV2 cell activation,which induced by GABA.Conclusions: Chronic cold exposure induced an increase in the level of GABA in mice.Excessive GABA induced the activation of microglia in the hippocampus through the NLRP3 inflammasome and NF-?B signaling pathway,released inflammatory cytokines,ultimately led to the damage of the neurons in hippocampus.