| Parkinson’s disease(PD)is a common neurodegenerative disease in the seniors,The characterized of the disease is the death of dopaminergic neurons.Even PD etiology is not clear,Scientists found that activation of microglia mediated nerve inflammation may lead to PD.Depression is a global mental illness,and the incidence rate is rising.Currently study suggested that chronic CUMS may cause depression.In addition,Studies have found that patients with depression are more likely to suffer from PD.Biochanin A can inhibit the activation of microglia,but it is not clear in PD model combined depression model.Objective:To investigate whether corticosterone pretreated could reinforce LPS activated BV2 cell.To investigate whether Bio A could inhibite BV2 cell activation and the the mechanism of the process.Methods:1.Using MTT assay to seek the appropriate dose of LPS.The conventional cultured BV2 cells were divided into 6 groups:(1)Control group;(2)LPS(0.01μg/ml)group;(3)LPS(0.1μg/ml)group;(4)LPS(1μg/ml)group;(5)LPS(10μg/ml)group;(6)LPS(100μg/ml)group.All groups were added the preset LPS incubated 36 h.2.Using MTT assay to seek the appropriate dose of Bio A.The conventional cultured BV2 cells were divided into 6 groups:(1)Control group;(2)Bio A(2.5μM)group;(3)Bio A(5μM)group;(4)Bio A(10μM)group;(5)Bio A(20μM)group;(6)Bio A(40μM)group.All groups were added the preset Bio A incubated 36 h.3.Using Griess assay to detect the nitric oxide of all groups for seeking the appropriate dose of CORT.The conventional cultured BV2 cells were divided into 7 groups:(1)Control group;(2)LPS(1μg/ml)group;(3)CORT(25n M)+ LPS(1μg/ml)group;(4)CORT(50n M)+ LPS(1μg/ml)group;(5)CORT(100n M)+ LPS(1μg/ml)group group;(6)CORT(200n M)+ LPS(1μg/ml)group;(7 CORT(400n M)+ LPS(1μg/ml)group).All groups except control were added CORT pretreated 2h,Then incubated 36 h with LPS.4.Using Griess assay to detect the nitric oxide of all groups.The conventional cultured BV2 cells were divided into 5 groups:(1)Control group;(2)CORT(50n M)group;(3)LPS(1μg/ml)group;(4)LPS(1μg/ml)+CORT(50n M)group;(5)LPS(1μg/ml)+CORT(50n M)+Bio A(5μM)group.All groups except control were added CORT pretreated2 h,Then incubated 36 h with LPS and Bio A.5.Using microscopic observe and DCFH-DA probe method to detect the effection of corticosterone pretreatment and the inhabitation of Bio A.The conventional cultured BV2 cells were divided into 5 groups:(1)Control group;(2)CORT(50n M)group;(3)LPS(1μg/ml)group;(4)LPS(1μg/ml)+CORT(50n M)group;(5)LPS(1μg/ml)+CORT(50n M)+Bio A(5μM)group.All groups except control were added CORT pretreated2 h,Then incubated 36 h with LPS and Bio A.6.Using Western-Blot to detect the expression of 5-HTT.The conventional cultured BV2 cells were divided into 5 groups:(1)Control group;(2)CORT(50n M)group;(3)LPS(10μg/ml)group;(4)LPS(1μg/ml)+CORT(50n M)group;(5)LPS(1μg/ml)+CORT(50n M)+Bio A(5μM)group.All groups except control were added CORT pretreated2 h,Then incubated 36 h with LPS and Bio A.7.Using Western-Blot to detect the expression of GR and MR.The conventional cultured BV2 cells were divided into 5 groups:(1)Control group;(2)CORT(50n M)group;(3)LPS(10μg/ml)group;(4)LPS(1μg/ml)+CORT(50n M)group;(5)LPS(1μg/ml)+CORT(50n M)+Bio A(5μM)group.All groups except control were added CORT pretreated 2h,Then incubated 36 h with LPS and Bio A.8.Using Western-Blot to detect the expression of TNF-α,IL-1β,IL-6.The conventional cultured BV2 cells were divided into 5 groups:(1)Control group;(2)CORT(50n M)group;(3)LPS(10μg/ml)group;(4)LPS(1μg/ml)+CORT(50n M)group;(5)LPS(1μg/ml)+CORT(50n M)+Bio A(5μM)group.All groups except control were added CORT pretreated 2h,Then incubated 36 h with LPS and Bio A.9.Using Western-Blot to detect the expression of ASC,caspase-1,NLRP3.The conventional cultured BV2 cells were divided into 5 groups:(1)Control group;(2)CORT(50n M)group;(3)LPS(10μg/ml)group;(4)LPS(1μg/ml)+CORT(50n M)group;(5)LPS(1μg/ml)+CORT(50n M)+Bio A(5μM)group.All groups except control were added CORT pretreated 2h,Then incubated 36 h with LPS and Bio A.Results:1.The dose of LPS more than 1μg/ml will induce cell apoptosis.so the dose of1 μg/ml is the ideal model concentration.2.When the dose of Bio A was more than 10 μM,it showed cytotoxicity,and the toxicity was more obviously at the dose of 20 μM.The dose of 5 μM was suitable to our experiment.3.Compared with control group,the CORT(50Nm)+LPS(1 μg/ml)group shows the most NO.3.Compared with control group,the LPS(1 μg/ml)group shows more NO;compared with LPS(1 μg/ml)group,the CORT(50n M)+ LPS(1 μg/ml)goroup shows more NO;compared with CORT(50n M)+ LPS(1 μg/ml)group,the CORT(50n M)+ LPS(1μg/ml)+Bio A(5μM)group shows less NO.4.Compared with control group,the LPS(1 μg/ml)group shows more ROS;Compared with LPS(1 μg/ml)group,the CORT(50n M)+ LPS(1 μg/ml)group shows more ROS;Compared with CORT(50n M)+ LPS(1 μg/ml)group,the CORT(50n M)+ LPS(1 μg/ml)+Bio A(5μM)group shows less ROS.6.Compared with control group,the 5-HTT expression of LPS(1 μg/ml)group was significantly decreased;Compared with LPS(1 μg/ml)group,the 5-HTT expression of CORT(50n M)+ LPS(1 μg/ml)group was significantly decreased;Compared with CORT(50n M)+ LPS(1 μg/ml)group,the 5-HTT expression of CORT(50n M)+ LPS(1 μg/ml)+Bio A(5μM)group was significantly increased.7.Compared with control group,the GR expression of LPS(1 μg/ml)group was significantly decreased;Compared with LPS(1 μg/ml)group,the GR expression of CORT(50n M)+ LPS(1 μg/ml)group was significantly decreased;Compared with CORT(50n M)+ LPS(1 μg/ml)group,the GR expression of CORT(50n M)+ LPS(1μg/ml)+Bio A(5μM)group was significantly increased.Compared with results of the GR,the expression MR show opposite.8.Compared with control group,the TNF-α,IL-1β,IL-6 expression of LPS(1 μg/ml)group was significantly increased;Compared with LPS(1 μg/ml)group,the TNF-α,IL-1β,IL-6 expression of CORT(50n M)+ LPS(1 μg/ml)group was significantly increased;Compared with CORT(50n M)+ LPS(1 μg/ml)group,the TNF-α,IL-1β,IL-6 expression of CORT(50n M)+ LPS(1 μg/ml)+Bio A(5μM)group was significantly decreased.9.Compared with control group,the ASC,caspase-1,NLRP3 expression of LPS(1 μg/ml)group was significantly increased;Compared with LPS(1 μg/ml)group,the ASC,caspase-1,NLRP3 expression of CORT(50n M)+ LPS(1 μg/ml)group was significantly increased;Compared with CORT(50n M)+ LPS(1 μg/ml)group,the ASC,caspase-1,NLRP3 expression of CORT(50n M)+ LPS(1 μg/ml)+Bio A(5μM)group was significantly decreased.Conclusion:1.Low dose of corticosterone pretreated reinforce LPS activated BV2 cells.2.Bio A can inhibite BV2 activation may via NLRP3 inflammasome. |
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