The Role Of Pyroptosis In The Pathogenesis Of SD Rat Kernicterus Model

Objective: The present study aims to investigate whether pyroptosis was involved in the pathogenesis of rat kernicterus model and through regulating the UCB-induced pyroptosis to observe neurological manifestation.To study the role of caspase1 activation in UCB-induced rat hippocampol neurons and the effect of VX-765 intervention on bilirubin neurontoxicity.Methods: 5-day-old SD rats were injected UCB solution into the cisterna magna to establish kernicterus model.The expression of NLRP3 and caspase1 were detected by Western blot.The effects of VX-765(caspase1 specific inhibitor)were evaluated,including the expression of caspase1,morphologic changes of nerve cells,cytokine production,general conditions,and behavioral tests(neurological evaluation described by Garcia et al,rotarod test,open field test and Morris water maze).Primary-cultured rat hippocampal neurons were randomly divided into control group,UCB group and VX-765 intervention group.The expression of NLRP3 and caspase1 were detected by Western blot,the relative cellsurvival and mortality were assessed with a modified MTT assay,Lactate dehydrogenase assay and Typan blue staining.IL-18 concentration in the supernatant was measured by ELISA.Results: Caspase1 was activated in the cerebrum at 7d after the kernicterus model was established,whereas,intraperitoneal administration of VX-765 could inhibit caspase1 activation,confirmed by Western blotting.Compared with UCB and vehicle+UCB groups,the nerve cells in VX-765-treated group showed better preserved and displayed less DNA fragmentation,furthermore,VX-765 could significantly reduce the release of IL-1? and IL-18.More importantly,not only the general conditions of the VX-765-treated rat were improved but also VX-765+UCB rats performed much better on long term neurological manifestation.In primary-cultured rat hippocampal neurons,UCB exposure for 3h and 6h,the expression of NLRP3 and activated caspase1 were significantly higher than the control group(3h: NLRP3 p=0.0005 caspase1 p=0.0122,6h: NLRP3 p<0.001 caspase1 p<0.001).While,VX-765 intervention could suppressed the activation of caspase1 compared with bilirubin group(3h p=0.0063,6h p=0.002).The relative survival rate in VX-765 intervention group was 84.020±2.311%,significantly higher than bilirubin group(p<0.001),but lower than control group(p<0.001),whereas,the release of LDH in VX-765 intervention group was 10.780±1.577%,significantly lower than bilirubin group(p<0.001),but higher than control group(p=0.0005).The mortality in VX-765 intervention group was5.580±1.234%,significantly lower than bilirubin group(p=0.0006),but higher than control group(p=0.0424).Conclusion: This study for the first time certifies that pyroptosis is involved in the process of rat kernicterus model.In addition,inhibition of the UCB-induced pyroptosis by intraperitoneal administration of VX-765 has strong neuroprotective effects.