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* * Astrocyte Reactivity to Unconjugated Bilirubin Requires TNF-α and IL-1β Receptor Signaling Pathways GLIA 59:14–25 (2011) SCI(2010):5.19 周康康 2012-7-2 1 We have reported that tumor necrosis factor (TNF)-α and interleukin (IL)-1β are produced by cultured neurons and mainly by glial cells exposed to unconjugated bilirubin (UCB). The effects of these cytokines are mediated by cell surface receptors through a nuclear factor (NF)-κB-dependent pathway that we have showed to be activated by UCB. Summary 2 Exposure of astrocytes to UCB increased the expression of both TNF- α receptor TNFR1 and IL-1 β receptor IL-1R1, but not TNFR2, as well as their activation, observed by augmented binding of receptors’ molecular adaptors, TRAF2 and TRAF6, respectively. 3 Silencing of TNFR1, using siRNA technology, or blockade of IL-1 β cascade, using its endogenous antagonist, IL-1 receptor antagonist (IL-1ra), prevented UCB-induced cytokine release and NF-κ B activation. 4 Interestingly, lack of TNF-α signal transduction reduced UCB-induced cell death for short periods of incubation, in contrast, inhibition of IL-1 β cascade produced a sustained blockade of astrocyte injury by UCB. 5 Together, our data show that inflammatory pathways are activated during in vitro exposure of rat astrocytes to UCB. This supports the concept that inflammatory pathways play a role in brain damage by UCB, and that they may represent important pharmacological targets. Materials and methods 1 Primary Culture of Astrocytes :2-day-old Wistar rats 2 Transient Transfection :three different doublestrand ed rat TNFR1 small interfering (si)RNAs (30 n M), scrambled siRNA (negative control) or the absence of siRNA (mock control). 3 Cell Treatment :50μM UCB plus 100μM human serum albumin (HSA) (UCB to HSA molar ratio of 0.5), from15 min to 24 h, at 370C. 4 Western Blot :The protein expression of TNFR1, TNFR2, and IL-1R1were determined by Western blot analysis. 5 Measurement
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