The Role Of A1 Astrocytes In Bilirubin-induced Microglia Injury

Objective: To investigate whether NF-κB and caspase-1 are involved in bilirubin-induced activation of A1-reactive astrocytes,and whether activated A1-reactive astrocytes may interact with other nerve cells.Methods: Primary cultured rat cortical astrocytes were divided into control group,bilirubin group,TAT-NBD group,and VX-765 group.The modified MTT method was used to detect the relative survival rate of cells,immunofluorescence assay was used to identify the expression of complement C3,MAP2 and NF-κB into the nucleus.Fluorescence quantitative PCR was used to detect the expression of C3,IL-1β,IL-18,Gpc4,Gpc6,Sparcl1,Thbs1,Thbs2,Mertk,Axl and Gas6 mRNA,Western blot was used to detect the expression of C3,NLRP3,activated caspase-1,and GSDMD-N.Results: The relative survival rate of astrocytes after bilirubin stimulation decreased with time,and the C3 mRNA level increased significantly at 6h and reached the peak at 12h(P < 0.01).The mRNA expressions of Gpc4,Gpc6,Sparcl1,Thbs1,Thbs2,Mertk,and Gas6 related to synaptic and phagocytic functions were significantly lower than those in the control group.Confocal microscopy showed that NF-κB was activated at 1h after bilirubin intervention,and peaked at 12 h with intracytoplasmic accumulation of C3.NLRP3 protein began to increase at3 h after bilirubin stimulation,and reached its peak at 6h(P < 0.05).Activated caspase-1 protein peaked at 6h and continued to 12h(P < 0.05).Gasdermin D-N-terminal(GSDMD-N)protein peaked at 6h(P < 0.05).After bilirubin stimulation,IL-1β mRNA was significantly increased at 3h and reached the peak at 6h(P < 0.01),while IL-18 mRNA also reached the peak at 6h(P < 0.05).After TAT-NBD and VX-765 intervention,the mRNA levels of C3 were decreased compared with bilirubin group(P < 0.05),the protein expressions of NLRP3,Caspase-1 and GSDMD-N were decreased(P < 0.05),IL-1β and IL-18 mRNA were decreased(P < 0.05),and the relative survival rate of astrocytes increased.After intervention with conditioned medium collected from UCB-treated strocytes,the relative survival rate of neurons decreased(P < 0.01),and the IL-1β mRNA level increased(P < 0.05).However,the mRNA levels of C3 and IL-1β in astrocytes were significantly increased after the intervention of MCM with bilirubin(P < 0.01).Conclusion: Bilirubin can induce activation of A1 reactive astrocytes.Inhibition of NF-κB and Caspase-1 can reduce activation of A1 reactive astrocytes induced by UCB,and exert anti-inflammatory protective effect.Meanwhile,the activation of A1 reactive astrocytes promoting neuronal death may be related to the interaction of microglia.