| Glucocorticoids(GCs)are secreted from the adrenal gland in response to activation of the hypothalamic-pituitary-adrenal(HPA)axis in a circadian pattern under basal conditions and are elevated as a part of the physiological responses to stressful events.GCs are also widely prescribed for treatment of autoimmune disease,excessive inflammation,allergies,and transplant rejection.However,a long term therapy with GCs have been linked to memory impairment,anxiety disorders,depression,and Alzheimer’s disease(AD).However,the mechanisms for GCs-induced neurodegeneration have not been fully elucidated.Part one ObjectiveTo observe the effects and mechanism of chronic glucocorticoids exposure on NLRP-1inflammasome activation in primary culture hippocampal neurons,and determine the role of NLRP-1 inflammasome in GCs-induced neurons injury.Methods1.Cultured hippocampal neurons in 5 days(d)were randomly divided into 6 groups:groups of control for 1d,3d and 5d,and groups of dexamethasone(DEX,5 μM)exposure for 1d,3d,5d.The neuronal damage was evaluated by measuring the amount of cytoplasmic lactae dehydrogenase(LDH)released in the supernatants of hippocampal neurons.The Hoechst 33258 staining was used to observe neuronal apoptosis.Western blot was used to observe the expression level of NLRP-1,ASC,Caspase-1 and IL-1β.ELISA was used to detect the expressions of IL-1β and IL-18.2.Cultured hippocampal neurons in 5 days were randomly divided into 6 groups:control group,DEX(0.1,1 and 5μM)group,RU486(5μM)group and DEX(5μM)+RU486(5μM)group for 3d or 5d.The neuronal damage was detected by measuring the amount of cytoplasmic lactae dehydrogenase(LDH)released in the supernatants of hippocampal neurons.Hoechst 33258 staining was used to observe neuronal apoptosis.Immunofluorescence was used to detect the expression of GR and MAP2.Western blot was used to observe the expression level of GR,NLRP-1,ASC,Caspase-1,IL-1β and NF-κB.ELISA was used to detect the expressions of IL-1β and IL-18.Q-PCR was used to detect the mRNA levels of NLRP-1,ASC,Caspase-1 and IL-1β.Result1.Compared with control group,DEX 5μM exposure for 3d and 5d could significantly increase the LDH release in the supernatants.Hochest 33258 staining results showed that DEX 5μM exposure for 1d,3d and 5d significantly increased the hippocampal neurons apoptosis.DEX 5μM exposure for 5 days significantly decreased the expression of MAP2.Western blot results showed that DEX 5μM exposure for 1d and 3d significantly increased the expression of NLRP-1,caspase-1 and IL-1β.2.Compared with control group,DEX 1 and 5μM exposure could significantly increase LDH in the supernatants,while RU486 could decrease LDH release induced by DEX exposure.Immunofluorescence results showed that DEX 1μM and 5μM exposure for 5 days significantly decreased the expression of MAP2;DEX 0.1,1 and 5μM exposure for 3d significantly decreased the expression of GR in the hippocampal neurons.Western blot results showed that DEX 0.1,1 and 5μM exposure for 3d significantly decreased the expression of GR;DEX(0.1,1,5μM)exposure for 3d significantly increased the expression of NLRP-1 and ASC,DEX 0.1μM exposure for 3d significantly decreased the expression of caspase-1 and IL-1β,and DEX 5μM exposure for 3d significantly increased the expression of caspase-1 and IL-1β.DEX 1μM and 5μM exposure for 3d significantly decreased the expression of NF-κB.Compared with DEX5μM treated group,RU486 significantly decreased the expression of caspase-1 and IL-1β.ELISA results showed that DEX 1 and 5μM exposure for 3d significantly increased the amount of IL-1β and IL-18 in the supernatants.Compared with DEX 5μM treated group,RU486 significantly decreased the release of IL-1β and IL-18.Part two ObjectiveTo observe the effects and mechanism of chronic glucocorticoids exposure on BK-NLRP1 signal activation in cortical and hippocampal neurons,and determine the role of BK-NLRP1 signal in GCs-induced neurons injury in hippocampal neurons,which provides new ideas and targets for prevention and treatment of AD.Methods1.Cultured hippocampal neurons in 5 days were randomly divided into 4 groups:control group,DEX(5μM)group,IbTx(IbTx,0.2μM)and DEX(5μM)+ IbTx(0.2μM)group for 3d.The cell damage was detected by measuring the amount of cytoplasmic lactae dehydrogenase(LDH)released in the supernatants of hippocampal neurons.Hoechst 33258 staining was used to observe neuronal apoptosis.Immunofluorescence was used to detect the expression of MAP2.Western blot was used to observe the expression level of MAP2,BK,NLRP-1,ASC,Caspase-1 and IL-1β.Q-PCR was used to detect the mRNA levels of NLRP-1,ASC,Caspase-1 and IL-1β.Whole cell patch clamp recording was used to observe large conductance calcium activated potassium channel channel(BK)currents.Ion-selective microelectrode method was used to detect intracellular K+concentration.ELISA was used to detect the expressions of IL-1β and IL-18.2.The male mice were divided into control group and DEX 5mg/kg group,each group was further divided into four time points of 7,14,21,28 d.Western blot and Q-PCR were used to detecte the m RNA and protein expression of BK.Result1.Compared with control group,DEX 5μM exposure for 3d significantly increased the expression of BK NLRP-1,ASC,Caspase-1,and IL-1β.Compared with DEX treated group,IbTx significantly decreased the LDH release increased by DEX exposure;IbTx also had a trend to decrease neuronal apoptosis.Western blot results showed that compared with DEX 5μM treated group,IbTx significantly reduced the expression of NLRP1,ASC,caspase-1 and IL-1β in the hippocampal neurons.ELISA results showed that compared with DEX 5μM treated group,IbTx significantly decreased the release of IL-1β and IL-18.The whole cell patch clamp results showed that DEX can increase BK currents and decreased [K]i in the hippocampal neurons,IbTx can inhibited DEX induced increased of BK currents and decrease in [K]i.ELISA results showed that compared with DEX 5μM treated group,IbTx significantly decreased the release of IL-1β and IL-18.2.Compared with control group,Q-PCR results showed that DEX 5mg/kg exposure for 21 d and 28 d significantly increase the expression of BK mRNA;Western blot results showed DEX 5mg/kg exposure for 7d,21 d and 28 d significantly increase the expression of BK.ConclusionChronic GCs exposure may down-regulate GR and increase brain inflammation via NLRP-1 inflammasome activation and induce hippocampal neurons damage.The hippocampal neuronal damage may be associated with the lower [K+]i induced by up-regulation and activation of BK channel in the hippocampal neurons.Chronic GCs exposure may increase neuroinflammation via activation of BK-NLRP1 signal pathway and promote hippocampal neuronal damage. |
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