astrocyte(星形胶质细胞)
Summary
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.
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.
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5 Measurement of Cytokine Release ：TNF-α, IL-1β, and IL-6 with specific DuoSetR ELISA Development kits.
6 Detection of NF-κB Activation — immunofluorescence detection ：rabbit anti-p65 NF- κB subunit antibody (1:200) as the primary antibodies, a FITC-labeled goat anti-rabbit antibody (1:160) as the secondary antibodies.
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Results
1 UCB Increases the Protein Content of TNFR1 and IL-1R1, but not of TNFR2, and Induces Their ement.
TNF-αcascade is mediated through the activation of two surface receptors, TNFR1 and TNFR2, while IL-1 β 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.
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.
7 Evaluation of Cell Death — LDH Astrocytes 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）
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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.
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.
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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).
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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.