Astrocyte Reactivity to Unconjugated Bilirubin Requires TNF- and IL-1 Receptor Signaling PathwaysGLIA 59:1425 (2011) SCI(2010)：5.19周康康 2012-7-211 We have reported that tumor necrosis factor (TNF)- and interleukin (IL)-1 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)-B-dependent pathway that we have showed to be activated by UCB. Summary22 Exposure of astrocytes to UCB increased the expression of both TNF- receptor TNFR1 and IL-1 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 cascade, using its endogenous antagonist, IL-1 receptor antagonist (IL-1ra), prevented UCB-induced cytokine release and NF- B activation.34 Interestingly, lack of TNF- signal transduction reduced UCB-induced cell death for short periods of incubation, in contrast, inhibition of IL-1 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.4Materials and methods1 Primary Culture of Astrocytes ：2-day-old Wistar rats2 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 ：50M UCB plus 100M humanserum 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.55 Measurement of Cytokine Release ：TNF-, IL-1, and IL-6 with specific DuoSetR ELISA Development kits.6 Detection of NF-B Activation immunofluorescence detection ：rabbit anti-p65 NF- B subunit antibody (1:200) as the primary antibodies, a FITC-labeled goat anti-rabbit antibody (1:160) as the secondary antibodies.7 Evaluation of Cell Death LDHAstrocytes were then identified in fixed cells by an antibody directed against GFAP.（神经胶质原纤维酸性蛋白）To identify the total number of cells, astroglial nuclei were stained with Hoechst dye 33258.（烟酸己可碱 DNA染料）6Results1 UCB Increases the Protein Content of TNFR1 and IL-1R1, but not of TNFR2, and Induces Their Engagement.TNF-cascade is mediated through the activation of two surface receptors, TNFR1 and TNFR2, while IL-1 pathway occurs via IL-1R 1 engagement. Engagement of cell surface receptors is followed by recruitment of several adaptor proteins to the receptor complex. TRAF2 and TRAF6.789These data indicate that astrocytes exposed to UCB preferentially express TNFR1 rather than TNFR2, at least during the first 24h of treatment, suggesting that TNF- action in our study model occurs essentially through TNFR1. UCB incubation significantly increased this effect with maximum levels at 1 h for TRAF2 /TNFR1 complex and lasting from 1 to 12 h for TRAF6 /IL-1R1 complex, indicating that UCB treatment increases the transduction of TNF- and IL-1 signals through TRAF2 and TRAF6, respectively.102 Transfection of TNFR1 siRNAs Silences TNFR1 Expression Elicited by UCB, While IL-1raPrevents IL-1R1 Engagement Triggered by UCB.1112133 Silencing of TNFR1 and Suppression of IL-1R1 Activity Reduces UCB-Induced Secretion of TNF- , IL-1 and IL-6.1415164 Silencing of TNFR1 and Suppression ofIL-1R1 Activity Reduces UCB-Induced Activation of NF-B.1718195 Silencing of TNFR1 and Suppression of IL-1R1 Activity Modulates UCB-Induced Cytotoxicity.2021222324Together, our results demonstrate that upon UCB exposure astrocytes mount an inflammatory response that is exacerbated and perpetuated in time by the activation of TNFR1 and IL-1R1 signaling pathways.Astrocyte Reactivity to Unconjugated Bilirubin Requires TNF- and IL-1 Receptor Signaling Pathways.25Hence, cytokine pathways should be taken in consideration when treating UCB-induced neurological dysfunction, emerging as new targets for drug intervention in the prevention of potential brain deficits resulting from neonatal jaundice.26