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Fig. 3 | Journal of Neuroinflammation

Fig. 3

From: Unconjugated bilirubin induces pyroptosis in cultured rat cortical astrocytes

Fig. 3

Caspase-1 activation involved in UCB-induced plasma membrane rupture. a Rat cortical astrocytes were treated as previously described for the indicated time periods. The supernatant was collected for the determination of released LDH activity. *p < 0.05, versus the control group (0 h); **p < 0.01, versus the control group (0 h) using a two-tailed Student’s t test with Dunnett’s test. b LDH released into the supernatant by a cell loss of membrane integrity at 24 h was quantified. *p < 0.05, versus the control group; #p < 0.05, versus the UCB group using one-way ANOVA with Bonferroni’s post hoc test. Four independent experiments were performed in duplicate. c The cells positive of trypan blue staining in different groups were counted at 24 h after stimulation. ***p < 0.001, versus the VX-765 group; ###p < 0.001, versus the UCB group using one-way ANOVA with Bonferroni’s post hoc test. After treatment, the cells were stained with the membrane-permeable dye Hoechst 33342 (blue) and the membrane-impermeant dyes (red), EtBr (MW 394) or EthD2 (MW 1293). Adherent cells were visualized by fluorescence microscopy (× 40 objective). Representative images are shown. The cells in the control group excluded both EtBr (d) and EthD2 (h). As a positive control, cells treated with Triton X-100 caused the uptake of EtBr (g) and EthD2 (k). Cells cultured with UCB showed a higher influx of EtBr (e) and a smaller influx of EthD2 (i). The caspase-1 inhibitor, VX-765, prevented astrocytic EtBr uptake (f) but had no significant effect on EthD2 uptake (j). The positive cells (red) were determined by randomly counting a minimum of four fields from two different coverslips per sample and expressed as a percentage of the total nucleus population (l). The results were representative of five independent experiments. Error bars, SD

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