Unconjugated Bilirubin Induces Pyroptosis In Cultured Rat Cortical Astrocytes

ObjectiveTo investigate whether a highly inflammatory form of cell death,pyroptosis,is contributed to bilirubin neurotoxicity on cultured rat cortical astrocytes.To study the effect of VX-765(caspase1 specific inhibitor)on bilirubin neurotoxicity in kernicterus model.MethodsCultured rat cortical astrocytes were randomly divided into control group,bilirubin group and VX-765 intervention group.The activity of caspase1 and the protein of NLRP3 were detected by Western blot.Further,VX-765,a potent and selective competitive drug,was used to inhibit the activation of caspase1.The effective of VX-765 in the astrocytes treated with bilirubin were observed,including the cell viability,the morphologic changes of the cell membrane and nucleus,and pro-inflammation cytokines production.Kernicterus model was established.The activity of caspase1 was assessed by Western blot,the content of IL-1?in homogenate of rats brain tissue was tested by ELISA and the expression of GFAP protein was detected by immunoflurescence.Further,VX-765 was administered intraperitoneally at 24 h and 1h before model established.Typical neurological manifestations and bodyweight were dynamically recorded.ResultsStimulation the astrocytes with unconjugated bilirubin(UCB)at conditions mimicking those of jaundiced newborn significantly increased the activation of caspase1.Further,caspase1 activity was inhibited by treatment with VX-765.The relative cell viability of VX-765 treated astrocytes was improved;meanwhile,formation of plasma membrane pores was prevented,as measured by lactate dehydrogenase release and EtBr uptake than UCB treated astrocytes.Moreover,the DNA fragment was partly attenuated and the release of IL-1? and IL-18 were apparently decreased.In kernicterus model,caspase1 activity and the content of IL-1?were significantly higher at 12 h after bilirubin injection compared with the control group(P <0.05 and P<0.01).Meanwile,the expression of GFAP protein in rat cortical was markedly increased(P<0.05).Treatment with VX-765 at 1h before model established showed strong neuroprotective effects,clinical manifestation scores was significantly lower and bodyweight gain was markedly increased compared with UCB group(P<0.01 and P <0.05).ConclusionOur study suggested that pyroptotic cell death modality was involved in process of bilirubin-induced neurological dysfunction during in vitro exposure of rat cortical astrocytes to UCB.In addition,inhibition the activation of caspase1 with VX-765 not only has strong neuro-protective effects in astrocytes under UCB challenge by preventing pyroptosis and alleviating inflammation in vitro,but also ameliorates the short-term prognosis of rats with kernicterus in vivo,which may throw light on a new strategy to protect developing brain against UCB neurotoxicity.