Abstract
BackgroundBilirubin-induced neurological dysfunction (BIND), a severe complication of extreme neonatal hyperbilirubinemia, could develop into permanent neurodevelopmental impairments. Several studies have demonstrated that inflammation and nerve cell death play important roles in bilirubin-induced neurotoxicity; however, the underlying mechanism remains unidentified.MethodsThe present study was intended to investigate whether pyroptosis, a highly inflammatory form of programmed cell death, participated in the bilirubin-mediated toxicity on cultured rat cortical astrocytes. Further, VX-765, a potent and selective competitive drug, was used to inhibit the activation of caspase-1. The effects of VX-765 on astrocytes treated with bilirubin, including the cell viability, morphological changes of the cell membrane and nucleus, and the production of pro-inflammation cytokines, were observed.ResultsStimulation of the astrocytes with unconjugated bilirubin (UCB) at the conditions mimicking those of jaundiced newborns significantly increased the activation of caspase-1. Further, caspase-1 activation was inhibited by treatment with VX-765. Compared with UCB-treated astrocytes, the relative cell viability of VX-765-pretreated astrocytes was improved; meanwhile, the formation of plasma membrane pores was prevented, as measured by lactate dehydrogenase release, trypan blue staining, and ethidium bromide (EtBr) uptake. Moreover, DNA fragmentation was partly attenuated and the release of IL-1β and IL-18 was apparently decreased.ConclusionPyroptosis is involved in the process of UCB-induced rat cortical astrocytes’ injury in vitro and may be the missing link of cell death and inflammatory response exacerbating UCB-related neurotoxicity. More importantly, the depression of caspase-1 activation, the core link of pyroptosis, attenuated UCB-induced cellular dysfunction and cytokine release, which might shed light on a new therapeutic approach to BIND.
Highlights
Bilirubin-induced neurological dysfunction (BIND), a severe complication of extreme neonatal hyperbilirubinemia, could develop into permanent neurodevelopmental impairments
unconjugated bilirubin (UCB) induced caspase-1 activation in cultured rat cortical astrocytes First, we used western blotting to investigate whether caspase-1 was activated in primary cultured astrocytes exposed to 50 μM UCB
The caspase-1 activation at 6 h (p = 0.0021, Fig. 1b) and 12 h (p < 0.001, Fig. 1c) was significantly inhibited in the VX-765 group compared with the UCB group
Summary
Bilirubin-induced neurological dysfunction (BIND), a severe complication of extreme neonatal hyperbilirubinemia, could develop into permanent neurodevelopmental impairments. Several studies have demonstrated that inflammation and nerve cell death play important roles in bilirubin-induced neurotoxicity; the underlying mechanism remains unidentified. Growing evidence has indicated that inflammation plays an important role in the physiological and pathological process of neurological dysfunction induced by UCB [5–7]. The activation of glial cells and the secretion of pro-inflammation cytokines are the characteristics of neuroinflammation [14]. The increased cytokines could further exacerbate nerve cell death and inflammatory responses by activating the MAPK and nuclear factor-kappaB (NF-κB) signaling cascades [17, 19, 20]. There may be a link between inflammation responses and nerve cell death caused by UCB, but the precise mechanism remains unclear
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